CN110461354A - protein conjugate - Google Patents

Abstract

Translated fromChinese

提供了精氨酸脱亚胺酶(ADI)和肿瘤坏死因子(TNF)超家族配体的缀合物，以及相关的组合物和其使用方法。还提供了六聚体多肽和三聚体多肽的缀合物、第一和第二三聚体多肽的缀合物以及相关的组合物和其使用方法。

Provided are conjugates of arginine deiminase (ADI) and tumor necrosis factor (TNF) superfamily ligands, and related compositions and methods of use thereof. Also provided are conjugates of hexameric polypeptides and trimeric polypeptides, conjugates of first and second trimeric polypeptides, and related compositions and methods of use thereof.

Description

Translated fromChinese

蛋白质缀合物protein conjugate

相关申请的交叉引用CROSS-REFERENCE TO RELATED APPLICATIONS

本申请根据35U.S.C.§119(e)要求于2017年3月29日提交的美国临时申请第62/478,398号的优先权，所述申请通过引用以其全文并入。This application claims priority under 35 U.S.C. § 119(e) to US Provisional Application No. 62/478,398, filed March 29, 2017, which is incorporated by reference in its entirety.

关于序列表的声明Statement Regarding Sequence Listing

与本申请相关的序列表以文本格式提供以代替纸质副本，并且在此通过引用并入本说明书中。含有序列表的文本文件的名称为TDWG_007_01WO_ST25.txt。所述文本文件为约251KB，于2018年3月28日创建，并通过EFS-Web以电子方式提交。The Sequence Listing related to this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into this specification. The text file containing the sequence listing is named TDWG_007_01WO_ST25.txt. Said text file is approximately 251KB, created on March 28, 2018, and submitted electronically via EFS-Web.

技术领域technical field

本公开部分涉及精氨酸脱亚胺酶(ADI)和肿瘤坏死因子(TNF)超家族配体的缀合物，以及相关的组合物和其使用方法。本公开还涉及六聚体多肽和三聚体多肽的缀合物、第一和第二三聚体多肽的缀合物以及相关的组合物和其使用方法。This disclosure relates, in part, to conjugates of arginine deiminase (ADI) and tumor necrosis factor (TNF) superfamily ligands, as well as related compositions and methods of use. The present disclosure also relates to conjugates of hexameric and trimeric polypeptides, conjugates of first and second trimeric polypeptides, and related compositions and methods of use thereof.

背景技术Background technique

精氨酸耗竭疗法可以有效治疗某些形式的癌症和其它疾病。例如，精氨酸脱亚胺酶可以用于通过将精氨酸转化为瓜氨酸和氨来耗尽精氨酸的血流供应。ADI-PEG 20是示例性ADI-PEG，目前临床上正在研究所述ADI-PEG以用于缺乏在将瓜氨酸转化为精氨酸中所涉及的关键酶精氨基琥珀酸合成酶-1(ASS1)的肿瘤。ADI-PEG 20具有良好的耐受性并且在临床研究中显示出前景(参见例如，Qiu等人，《癌症快报(Cancer Lett.)》，2015年8月1日；364(1):1-7；Phillips等人，《癌症研究和治疗(Cancer Res Treat.)》，2013年12月；45(4):251-62；Feun等人，《当前药物设计(Curr Pharm Des.)》，2008；14(11):1049-57；Feun和Savaraj，《调研药物专家评论(Expert Opin Investig Drugs.)》，2006年7月；15(7):815-22；Feun等人，《临床营养和代谢护理的最新观点(Curr Opin Clin Nutr Metab Care.)》，2015年1月；18(1):78-82)。Arginine depletion therapy can be effective in treating certain forms of cancer and other diseases. For example, arginine deiminase can be used to deplete the bloodstream supply of arginine by converting arginine to citrulline and ammonia. ADI-PEG 20 is an exemplary ADI-PEG that is currently being studied clinically for lack of argininosuccinate synthase-1 ( ASS1) tumors. ADI-PEG 20 is well tolerated and has shown promise in clinical studies (see eg, Qiu et al, Cancer Lett., 2015 Aug 1;364(1):1- 7; Phillips et al., Cancer Res Treat. 2013 Dec;45(4):251-62; Feun et al., Curr Pharm Des., 2008 ; 14(11):1049-57; Feun and Savaraj, Expert Opin Investig Drugs. 2006 Jul;15(7):815-22; Feun et al., Clinical Nutrition and Current Perspectives in Metabolic Care (Curr Opin Clin Nutr Metab Care. 2015 Jan;18(1):78-82).

通过合适的配体激活肿瘤坏死因子(TNF)受体超家族的细胞表面死亡受体代表一种有吸引力的通过癌细胞中的细胞凋亡诱导细胞死亡的治疗策略(参见例如，Palacios等人，《当前药物设计》，2014；20(17):2819-33)。作为一个实例，TNF相关的凋亡诱导配体(TRAIL，也称为Apo2L)具有在癌细胞中选择性诱导凋亡的能力，并且已在许多临床前研究中证明了强大的抗癌活性。Activation of cell surface death receptors of the tumor necrosis factor (TNF) receptor superfamily by appropriate ligands represents an attractive therapeutic strategy to induce cell death through apoptosis in cancer cells (see, eg, Palacios et al. , Current Drug Design, 2014;20(17):2819-33). As an example, TNF-related apoptosis-inducing ligand (TRAIL, also known as Apo2L) has the ability to selectively induce apoptosis in cancer cells and has demonstrated potent anticancer activity in many preclinical studies.

然而，仍然需要优化这些药剂和其它药剂的药代动力学和/或生物活性。本公开提供了这些益处和其它益处。However, there remains a need to optimize the pharmacokinetics and/or biological activities of these and other agents. The present disclosure provides these and other benefits.

发明内容SUMMARY OF THE INVENTION

本公开的实施例包含缀合物，其包括共价连接到肿瘤坏死因子(TNF)超家族配体的精氨酸脱亚胺酶(ADI)。Embodiments of the present disclosure include conjugates comprising arginine deiminase (ADI) covalently linked to a tumor necrosis factor (TNF) superfamily ligand.

在一些实施例中，所述ADI包括与选自表A1的序列至少90％、95％、96％、97％、98％或99％相同的氨基酸序列，由所述氨基酸序列组成或基本上由所述氨基酸序列组成。在一些实施例中，所述ADI是六聚体ADI多肽，例如，同源六聚体多肽。一些实施例中，所述六聚体或同源六聚体ADI包括与SEQ ID NO:9、37、38、50或57-68至少90％、95％、96％、97％、98％或99％相同的氨基酸序列，由所述氨基酸序列组成或基本上由所述氨基酸序列组成。In some embodiments, the ADI comprises, consists of, or consists essentially of an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from Table A1 The amino acid sequence composition. In some embodiments, the ADI is a hexameric ADI polypeptide, eg, a homohexameric polypeptide. In some embodiments, the hexameric or homohexameric ADI comprises at least 90%, 95%, 96%, 97%, 98% or Amino acid sequences that are 99% identical, consist of, or consist essentially of, said amino acid sequences.

在一些实施例中，所述TNF超家族配体选自表T1。在一些实施例中，所述超家族配体选自TNF相关的凋亡诱导配体(TRAIL)、TNF-α和FasL。在一些实施例中，所述TNF超家族配体包括与选自表T2的序列至少90％、95％、96％、97％、98％或99％相同的氨基酸序列，由所述氨基酸序列组成或基本上由所述氨基酸序列组成。在一些实施例中，所述TNF超家族配体是三聚体或同源三聚体多肽。In some embodiments, the TNF superfamily ligand is selected from Table T1. In some embodiments, the superfamily ligand is selected from the group consisting of TNF-related apoptosis-inducing ligand (TRAIL), TNF-alpha, and FasL. In some embodiments, the TNF superfamily ligand comprises, consists of, an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from Table T2 or consist essentially of said amino acid sequence. In some embodiments, the TNF superfamily ligand is a trimeric or homotrimeric polypeptide.

在一些实施例中，所述ADI和所述TNF超家族配体通过连接子，任选地生理稳定的连接子分开。在一些实施例中，所述连接子为肽连接子，任选地为柔性肽连接子或刚性肽连接子。在一些实施例中，所述肽连接子的长度为约1-100个氨基酸、约1-90个氨基酸、约1-80个氨基酸、约1-70个氨基酸、约1-80个氨基酸、约1-50个氨基酸、约1-40个氨基酸、约1-30个氨基酸、约1-20个氨基酸、约1-10个氨基酸或约1-5个氨基酸，或长度为约1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、60、70、80、90或100个氨基酸。在一些实施例中，所述肽连接子选自表L1。In some embodiments, the ADI and the TNF superfamily ligand are separated by a linker, optionally a physiologically stable linker. In some embodiments, the linker is a peptide linker, optionally a flexible peptide linker or a rigid peptide linker. In some embodiments, the peptide linker is about 1-100 amino acids, about 1-90 amino acids, about 1-80 amino acids, about 1-70 amino acids, about 1-80 amino acids, about 1-50 amino acids, about 1-40 amino acids, about 1-30 amino acids, about 1-20 amino acids, about 1-10 amino acids, or about 1-5 amino acids, or about 1, 2, 3 amino acids in length , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80 , 90 or 100 amino acids. In some embodiments, the peptide linker is selected from Table L1.

在一些实施例中，所述缀合物是融合多肽。在一些实施例中，所述ADI融合到TNF超家族配体的N端，任选地通过连接子分开。在一些实施例中，所述ADI融合到TNF超家族配体的C端，并任选地通过连接子分开。In some embodiments, the conjugate is a fusion polypeptide. In some embodiments, the ADI is fused to the N-terminus of a TNF superfamily ligand, optionally separated by a linker. In some embodiments, the ADI is fused to the C-terminus of a TNF superfamily ligand and is optionally separated by a linker.

在一些实施例中，所述连接子是非肽连接子。In some embodiments, the linker is a non-peptide linker.

在一些实施例中，所述缀合物相对于单独的所述ADI和/或单独的所述TNF超家族配体具有改善的药代动力学、物理和/或生物学性质，任选地选自增加的稳定性、增加的血清半衰期、提高的生物利用度、增加的生物活性、增加的暴露度和降低的清除率中的一种或多种。In some embodiments, the conjugate has improved pharmacokinetic, physical and/or biological properties relative to the ADI alone and/or the TNF superfamily ligand alone, optionally selected One or more of increased stability, increased serum half-life, increased bioavailability, increased biological activity, increased exposure, and decreased clearance.

在一些实施例中，述缀合物相对于单独的所述ADI和/或单独的所述TNF超家族配体具有增加的稳定性和/或血清半衰期，任选地其中与所述缀合物相关的所述稳定性和/或血清半衰期相对于单独的所述ADI和/或单独的所述TNF超家族配体增加了约或至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、200％、300％、400％、500％、600％、700％、800％、900％或1000％或更多。In some embodiments, the conjugate has increased stability and/or serum half-life relative to the ADI alone and/or the TNF superfamily ligand alone, optionally wherein the conjugate is The associated stability and/or serum half-life is increased by about or at least about 10%, 20%, 30%, 40%, 50% relative to the ADI alone and/or the TNF superfamily ligand alone , 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% or more.

在一些实施例中，所述缀合物相对于单独的所述ADI和/或单独的所述TNF超家族配体具有增加的生物活性，任选地其中所述缀合物的生物活性相对于单独的所述ADI和/或单独的所述TNF超家族配体增加了约或至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、200％、300％、400％、500％、600％、700％、800％、900％或1000％或更多，或任选地其中所述生物活性相对于单独的所述ADI和/或单独的所述TNF超家族配体协同增加。在一些实施例中，所述生物活性是在癌细胞中诱导细胞死亡或凋亡，所述诱导相对于单独的ADI和/或单独的TNF超家族配体任选地协同增加。In some embodiments, the conjugate has increased biological activity relative to the ADI alone and/or the TNF superfamily ligand alone, optionally wherein the biological activity of the conjugate is relative to Said ADI alone and/or said TNF superfamily ligand alone increases by about or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% or more, or optionally wherein said biological activity is relative to said ADI and and/or said TNF superfamily ligands alone are synergistically increased. In some embodiments, the biological activity is the induction of cell death or apoptosis in cancer cells, the induction optionally being synergistically increased relative to ADI alone and/or TNF superfamily ligand alone.

在一些实施例中，所述癌细胞是ADI敏感的细胞，其任选地选自乳腺癌细胞、肝细胞癌细胞、伯基特淋巴瘤细胞、结肠癌细胞、胶质母细胞瘤癌细胞、白血病细胞、黑色素瘤癌细胞、非小细胞肺癌(NSCLC)细胞、卵巢癌细胞、胰腺癌细胞、前列腺癌细胞和肾癌细胞中的一种或多种。In some embodiments, the cancer cells are ADI-sensitive cells optionally selected from breast cancer cells, hepatocellular carcinoma cells, Burkitt lymphoma cells, colon cancer cells, glioblastoma cancer cells, One or more of leukemia cells, melanoma cancer cells, non-small cell lung cancer (NSCLC) cells, ovarian cancer cells, pancreatic cancer cells, prostate cancer cells, and renal cancer cells.

在一些实施例中，所述癌细胞是ADI不敏感的细胞，其任选地选自乳腺癌细胞、结肠癌细胞和NSCLC细胞中的一种或多种。In some embodiments, the cancer cells are ADI-insensitive cells, optionally selected from one or more of breast cancer cells, colon cancer cells, and NSCLC cells.

在一些实施例中，相对于单独的所述TNF超家族配体，所述ADI任选地使所述TNF超家族配体诱导癌细胞中细胞死亡或凋亡的能力增加约至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、200％、300％、400％、500％、600％、700％、800％、900％或1000％。In some embodiments, the ADI optionally increases the ability of the TNF superfamily ligand to induce cell death or apoptosis in cancer cells by about at least about 10% relative to the TNF superfamily ligand alone, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000%.

在一些实施例中，所述ADI上调所述癌细胞上死亡受体5(DR5)的表达。In some embodiments, the ADI upregulates the expression of death receptor 5 (DR5) on the cancer cells.

在一些实施例中，所述ADI多肽通过连接基团共价键合到至少一个聚乙二醇(PEG)分子，任选地其中所述TNF超家族配体不共价键合到PEG分子。In some embodiments, the ADI polypeptide is covalently bonded to at least one polyethylene glycol (PEG) molecule through a linking group, optionally wherein the TNF superfamily ligand is not covalently bonded to the PEG molecule.

还包含缀合物，其包括(a)共价连接到三聚体多肽的六聚体多肽，或(b)共价连接到第二三聚体多肽的第一三聚体多肽，所述第二三聚体多肽与所述第一三聚体多肽不同。Also included are conjugates comprising (a) a hexameric polypeptide covalently linked to a trimeric polypeptide, or (b) a first trimeric polypeptide covalently linked to a second trimeric polypeptide, the The ditrimeric polypeptide is different from the first trimeric polypeptide.

在一些实施例中，所述六聚体多肽是同源六聚体多肽。在一些实施例中，所述六聚体多肽选自精氨酸脱亚胺酶，任选地如本文所述的以及脂联素或其胶原样结构域。In some embodiments, the hexameric polypeptide is a homohexameric polypeptide. In some embodiments, the hexameric polypeptide is selected from arginine deiminase, optionally as described herein, and adiponectin or a collagen-like domain thereof.

在一些实施例中，所述三聚体多肽是同源三聚体多肽。In some embodiments, the trimeric polypeptide is a homotrimeric polypeptide.

在一些实施例中，(b)的所述第一三聚体多肽选自脂联素或其胶原样结构域、T4次要纤维蛋白或其三聚结构域(折叠)、胶原的C-前肽、表面活性剂蛋白A(SP-A)和甘露糖结合蛋白A(MBP-A)。In some embodiments, the first trimeric polypeptide of (b) is selected from the group consisting of adiponectin or its collagen-like domain, T4 minor fibrin or its trimerization domain (fold), C-pro-collagen Peptides, Surfactant Protein A (SP-A) and Mannose Binding Protein A (MBP-A).

在一些实施例中，(a)的所述三聚体多肽或(b)的所述第二三聚体多肽选自肿瘤坏死因子(TNF)超家族配体，任选地如本文所述的。在一些实施例中，对于(a)，六聚体多肽共价连接到三聚体多肽的N端，或者对于(b)，第一三聚体多肽共价连接到第二三聚体多肽的N端。在一些实施例中，对于(a)，六聚体多肽共价连接到三聚体多肽的C端，或者其中对于(b)，第一三聚体多肽共价连接到第二三聚体多肽的C端。在一些实施例中，对于(a)，六聚体多肽和三聚体多肽通过连接子分开，或者对于(b)，第一和第二三聚体多肽通过连接子分开，其中所述连接子任选地为生理稳定的连接子。In some embodiments, the trimeric polypeptide of (a) or the second trimeric polypeptide of (b) is selected from tumor necrosis factor (TNF) superfamily ligands, optionally as described herein . In some embodiments, for (a) the hexameric polypeptide is covalently linked to the N-terminus of the trimeric polypeptide, or for (b) the first trimeric polypeptide is covalently linked to the second trimeric polypeptide N-terminal. In some embodiments, for (a), the hexameric polypeptide is covalently linked to the C-terminus of the trimeric polypeptide, or wherein for (b), the first trimeric polypeptide is covalently linked to the second trimeric polypeptide the C terminal. In some embodiments, for (a), the hexameric polypeptide and the trimeric polypeptide are separated by a linker, or for (b), the first and second trimeric polypeptides are separated by a linker, wherein the linker Optionally a physiologically stable linker.

在一些实施例中，所述缀合物是融合多肽。In some embodiments, the conjugate is a fusion polypeptide.

在一些实施例中，所述缀合物相对于单独的所述六聚体和/或三聚体多肽物理具有改善的药代动力学和/或生物学性质。在一些实施例中，对于(a)，与所述六聚体多肽缀合使所述三聚体多肽的稳定性和/或血清半衰期相对于单独的所述三聚体多肽任选地增加约或至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、200％、300％、400％、500％、600％、700％、800％、900％或1000％或更多，或者对于(b)，与所述第一三聚体多肽缀合使所述第二三聚体多肽的稳定性和/或血清半衰期相对于单独的所述第二三聚体多肽任选地增加约或至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、200％、300％、400％、500％、600％、700％、800％、900％或1000％或更多。In some embodiments, the conjugate physically has improved pharmacokinetic and/or biological properties relative to the hexameric and/or trimeric polypeptide alone. In some embodiments, for (a), conjugation to the hexameric polypeptide optionally increases the stability and/or serum half-life of the trimeric polypeptide relative to the trimeric polypeptide alone by about or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700% , 800%, 900%, or 1000% or more, or for (b), conjugation to the first trimeric polypeptide increases the stability and/or serum half-life of the second trimeric polypeptide relative to that alone of said second trimeric polypeptide is optionally increased by about or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% or more.

某些实施例涉及编码本文所述缀合物的分离的多核苷酸，其中所述缀合物是融合蛋白。还包含包括分离的多核苷酸的表达载体和包括分离的多核苷酸或表达载体的宿主细胞。Certain embodiments relate to isolated polynucleotides encoding the conjugates described herein, wherein the conjugates are fusion proteins. Also included are expression vectors comprising the isolated polynucleotides and host cells comprising the isolated polynucleotides or expression vectors.

还包含治疗组合物，其包括本文所述的缀合物和药学上可接受的载剂或赋形剂。在特定实施例中，缀合物形成六个ADI-TRAIL和/或TRAIL-ADI缀合物(任选地呈融合蛋白形式)的六聚体复合物(参见例如，图4)。在一些实施例中，缀合物或组合物的纯度为至少约95％且聚集度小于约5％，并且基本上不含内毒素。Also included are therapeutic compositions comprising the conjugates described herein and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the conjugate forms a hexameric complex of six ADI-TRAIL and/or TRAIL-ADI conjugates (optionally in the form of fusion proteins) (see eg, Figure 4). In some embodiments, the conjugate or composition is at least about 95% pure and has a degree of aggregation less than about 5%, and is substantially free of endotoxin.

还包括在有需要的受试者中治疗癌症、改善癌症症状或减少癌症进展的方法，包括对受试者施用本文所述的缀合物或治疗组合物。Also included are methods of treating cancer, ameliorating the symptoms of cancer, or reducing the progression of cancer in a subject in need thereof, comprising administering to the subject a conjugate or therapeutic composition described herein.

在一些实施例中，癌症选自以下中的一个或多个：肝细胞癌(HCC)、黑色素瘤、转移性黑色素瘤、胰腺癌、前列腺癌、小细胞肺癌、间皮瘤、淋巴细胞性白血病、慢性髓细胞性白血病、淋巴瘤、肝癌、肉瘤、白血病、急性髓性白血病、复发性急性髓性白血病、B细胞恶性肿瘤、乳腺癌、卵巢癌、结直肠癌、胃癌、胶质瘤(例如，星形细胞瘤、少突神经胶质瘤、室管膜瘤或脉络丛乳头状瘤)、多形性胶质母细胞瘤(例如，巨细胞胶质母细胞瘤或胶质肉瘤)、脑膜瘤、垂体腺瘤、前庭神经鞘瘤、原发性CNS淋巴瘤、原始神经外胚层肿瘤(成神经管细胞瘤)、非小细胞肺癌(NSCLC)、肾癌、膀胱癌、子宫癌、食道癌、脑癌、头颈癌、宫颈癌、睾丸癌和胃癌。In some embodiments, the cancer is selected from one or more of the following: hepatocellular carcinoma (HCC), melanoma, metastatic melanoma, pancreatic cancer, prostate cancer, small cell lung cancer, mesothelioma, lymphocytic leukemia , chronic myeloid leukemia, lymphoma, liver cancer, sarcoma, leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia, B cell malignancies, breast cancer, ovarian cancer, colorectal cancer, gastric cancer, glioma (e.g. , astrocytoma, oligodendroglioma, ependymoma, or choroid plexus papilloma), glioblastoma multiforme (eg, giant cell glioblastoma or gliosarcoma), meninges Tumor, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), non-small cell lung cancer (NSCLC), kidney cancer, bladder cancer, uterine cancer, esophageal cancer , brain, head and neck, cervical, testicular and gastric cancers.

附图说明Description of drawings

图1A-1D示出了与单独的每种药剂相比ADI-PEG 20和rhTRAIL对各种癌细胞系的相对活力的协同作用。Figures 1A-1D show the synergistic effect of ADI-PEG 20 and rhTRAIL on the relative viability of various cancer cell lines compared to each agent alone.

图2A-2C表明了与Raji Burkitt淋巴瘤细胞系中单独的每种药剂相比，ADI-PEG20和rhTRAIL对半胱天冬酶3/7活化(图2A)、细胞死亡诱导(图2B)以及未致力于细胞凋亡的活细胞百分比降低(半胱天冬酶3/7未活化的细胞；图2C)的协同作用。Figures 2A-2C show caspase 3/7 activation (Figure 2A), induction of cell death (Figure 2B) and induction of cell death (Figure 2B) by ADI-PEG20 and rhTRAIL compared to each agent alone in Raji Burkitt's lymphoma cell line. Synergistic effect of reduced percentage of viable cells not committed to apoptosis (caspase 3/7 inactivated cells; Figure 2C).

图3A-3D示出了用ADI-PEG 20处理后各种癌细胞系中TRAIL受体DR5的上调表达。Figures 3A-3D show up-regulated expression of the TRAIL receptor DR5 in various cancer cell lines following treatment with ADI-PEG 20.

图4表明了ADI敏感细胞系用ADI-PEG 20处理后存活蛋白水平降低。存活蛋白已被证明能阻止TRAIL的活性。因此，降低存活蛋白水平(连同DR5上调)可能有助于ADI增强和/或增加癌细胞系中TRAIL的凋亡活性的能力。Figure 4 shows that ADI-sensitive cell lines were treated with ADI-PEG 20 to reduce survivin levels. Survivin has been shown to block the activity of TRAIL. Therefore, reducing survivin levels (along with DR5 upregulation) may contribute to the ability of ADI to enhance and/or increase the apoptotic activity of TRAIL in cancer cell lines.

图5示出了由六个ADI-TRAIL和/或TRAIL-ADI缀合物，例如融合蛋白构成的六聚体复合物。Figure 5 shows a hexameric complex consisting of six ADI-TRAIL and/or TRAIL-ADI conjugates, eg fusion proteins.

图6A-6C示出了相对于单独的rhTRAIL、单独的M.col.ADI和作为单独多肽的rhTRAIL和M.col.ADI的组合，示例性M.col.ADI-TRAIL融合多肽与L1连接子(见表E3)对抗ADI的Colo 205癌细胞系中半胱天冬酶3/7诱导(图6A)和相对细胞活力(图6B和6C)的作用。Figures 6A-6C show exemplary M.col.ADI-TRAIL fusion polypeptides with the L1 linker relative to rhTRAIL alone, M.col.ADI alone, and a combination of rhTRAIL and M.col.ADI as individual polypeptides (See Table E3) Effects of caspase 3/7 induction (Fig. 6A) and relative cell viability (Figs. 6B and 6C) in ADI-resistant Colo 205 cancer cell lines.

图7A-7C示出了相对于单独的rhTRAIL、单独的M.col.ADI以及作为单独多肽的rhTRAIL和M.col.ADI的组合，示例性M.col.ADI-TRAIL融合多肽与L1连接子(见表E3)对ADI敏感的HCT116肿瘤细胞系中半胱天冬酶3/7诱导(图7A)和相对细胞活力(图7B和7C)的作用。Figures 7A-7C show exemplary M.col.ADI-TRAIL fusion polypeptides with an L1 linker relative to rhTRAIL alone, M.col.ADI alone, and a combination of rhTRAIL and M.col.ADI as individual polypeptides (See Table E3) Effects of caspase 3/7 induction (Fig. 7A) and relative cell viability (Figs. 7B and 7C) in the ADI-sensitive HCT116 tumor cell line.

图8A-8B示出了相对于单独的rhTRAIL、单独的M.col.ADI和作为单独多肽的rhTRAIL和M.col.ADI的组合，示例性M.col.ADI-TRAIL融合多肽与L1连接子(见表E3)对ADI敏感的Jurkat肿瘤细胞系中半胱天冬酶3/7诱导(图8A)和相对细胞活力(图8B)的作用。Figures 8A-8B show exemplary M.col.ADI-TRAIL fusion polypeptides with the L1 linker relative to rhTRAIL alone, M.col.ADI alone, and a combination of rhTRAIL and M.col.ADI as individual polypeptides (See Table E3) Effects of caspase 3/7 induction (FIG. 8A) and relative cell viability (FIG. 8B) in ADI-sensitive Jurkat tumor cell lines.

图9A-9D示出了来自表E1的示例性ADI-TRAIL融合多肽对抗ADI的Colo 205癌细胞系中半胱天冬酶3/7诱导(图9A和9B)和相对细胞活力(图9C和9D)的作用。Figures 9A-9D show caspase 3/7 induction (Figures 9A and 9B) and relative cell viability (Figures 9C and 9B) in the ADI-resistant Colo 205 cancer cell line of exemplary ADI-TRAIL fusion polypeptides from Table E1 9D).

图10A-10C示出了示例性ADI-TRAIL融合多肽对ADI敏感的HCT116细胞系中半胱天冬酶3/7诱导(图10A)和相对细胞活力(图10B-10C)的作用。Figures 10A-10C show the effect of exemplary ADI-TRAIL fusion polypeptides on caspase 3/7 induction (Figure 10A) and relative cell viability (Figures 10B-10C) in the ADI-sensitive HCT116 cell line.

图11A-11B示出了示例性ADI-TRAIL融合多肽对ADI敏感的Jurkat细胞系中半胱天冬酶3/7诱导(图11A)和相对细胞活力(图11B)的作用。Figures 11A-11B show the effect of exemplary ADI-TRAIL fusion polypeptides on caspase 3/7 induction (Figure 11A) and relative cell viability (Figure 11B) in ADI-sensitive Jurkat cell lines.

图12A-12C示出了在M.col.ADI(K192C或K287C)中具有点突变的示例性M.col.ADI-TRAIL融合多肽(包含非聚乙二醇化的和用2K或20K PEG聚乙二醇化的M.col.ADI-TRAIL融合多肽)对抗ADI的Colo 205细胞系中半胱天冬酶3/7诱导(图12A)和相对细胞活力(图12B-12C)的作用。Figures 12A-12C show exemplary M.col.ADI-TRAIL fusion polypeptides (comprising non-PEGylated and PEGylated with 2K or 20K PEG) with point mutations in M.col.ADI (K192C or K287C) The effect of diolated M.col. ADI-TRAIL fusion polypeptide) on caspase 3/7 induction (FIG. 12A) and relative cell viability (FIGS. 12B-12C) in ADI-resistant Colo 205 cell line.

图13A-13C示出了在M.col.ADI(K192C或K287C)中具有点突变的示例性M.col.ADI-TRAIL融合多肽(包含非聚乙二醇化的和用2K或20K PEG聚乙二醇化的M.col.ADI-TRAIL融合多肽)对ADI敏感的HCT116细胞系中半胱天冬酶3/7诱导(图13A)和相对细胞活力(图13B-13C)的作用。Figures 13A-13C show exemplary M.col.ADI-TRAIL fusion polypeptides (comprising non-PEGylated and PEGylated with 2K or 20K PEG) with point mutations in M.col.ADI (K192C or K287C) Effects of diolated M.col. ADI-TRAIL fusion polypeptide) on caspase 3/7 induction (FIG. 13A) and relative cell viability (FIGS. 13B-13C) in the ADI-sensitive HCT116 cell line.

图14A-14C示出了示例性TRAIL-M.col.ADI与M.col.ADI-TRAIL融合多肽对抗ADI的Colo 205细胞系中半胱天冬酶3/7诱导(图14A)和相对细胞活力(图14B-14C)的作用。Figures 14A-14C show caspase 3/7 induction (Figure 14A) and relative cells in ADI-resistant Colo 205 cell line with exemplary TRAIL-M.col.ADI and M.col.ADI-TRAIL fusion polypeptides The effect of viability (FIGS. 14B-14C).

图15A-15C示出了示例性TRAIL-M.col.ADI与M.col.ADI-TRAIL融合多肽对ADI敏感的HCT116细胞系中半胱天冬酶3/7诱导(图15A)和相对细胞活力(图15B-15C)的作用。Figures 15A-15C show caspase 3/7 induction (Figure 15A) and relative cells in the ADI-sensitive HCT116 cell line by exemplary TRAIL-M.col.ADI and M.col.ADI-TRAIL fusion polypeptides The effect of viability (FIGS. 15B-15C).

图16A-16B显示了静脉施用单剂量30mg/kg后，M.col.ADI-TRAIL在CD-1小鼠血清中随时间的药代动力学(PK)。在单次注射融合蛋白后，在CD-1小鼠的血清中测量M.col.ADI-TRAIL蛋白水平(图16A-16B)、精氨酸和瓜氨酸水平(图16A)以及针对融合蛋白M.col.ADI-TRAIL、M.col.ADI和rhTRAIL(通过ELISA评估，图16B)的抗体滴度。Figures 16A-16B show the pharmacokinetics (PK) of M.col. ADI-TRAIL in CD-1 mouse serum over time following intravenous administration of a single dose of 30 mg/kg. M.col. ADI-TRAIL protein levels (FIG. 16A-16B), arginine and citrulline levels (FIG. 16A) were measured in the serum of CD-1 mice following a single injection of the fusion protein and for fusion protein Antibody titers for M.col.ADI-TRAIL, M.col.ADI and rhTRAIL (assessed by ELISA, Figure 16B).

图17A-17F表明了M.col.ADI-TRAIL在HCT116异种移植模型中的功效。融合蛋白未引起任何明显的体重减轻(图17A)和肿瘤生长减少(图17B-17F)。*p<0.05，**p<0.01，***p<0.001。通过双向ANOVA评估了与媒剂处理对照组相比融合蛋白处理组中肿瘤减少的统计显著性。Figures 17A-17F demonstrate the efficacy of M.col. ADI-TRAIL in the HCT116 xenograft model. The fusion protein did not cause any significant weight loss (FIG. 17A) and tumor growth (FIGS. 17B-17F). *p<0.05, **p<0.01, ***p<0.001. Statistical significance of tumor reduction in the fusion protein-treated group compared to the vehicle-treated control group was assessed by two-way ANOVA.

图18A-18D示出血清ADI-TRAIL与肿瘤体积呈负相关(图18B-18C)。通过ELISA(总蛋白)和生物测定(活性蛋白)测量的融合蛋白浓度彼此相似。在肿瘤植入后的第21天和第28天，从荷瘤小鼠中提取血清。图17A-17F中示出了处理计划和肿瘤生长。Figures 18A-18D show that serum ADI-TRAIL is inversely correlated with tumor volume (Figures 18B-18C). The fusion protein concentrations measured by ELISA (total protein) and bioassay (active protein) were similar to each other. Serum was extracted from tumor-bearing mice on days 21 and 28 after tumor implantation. Treatment plans and tumor growth are shown in Figures 17A-17F.

图18D中示出了这些血清样品中的精氨酸和瓜氨酸水平。Arginine and citrulline levels in these serum samples are shown in Figure 18D.

图19表明了用M.col.ADI-TRAIL处理后HCT116异种移植模型中的剂量依赖性肿瘤生长减少。Figure 19 shows a dose-dependent reduction in tumor growth in a HCT116 xenograft model following treatment with M.col. ADI-TRAIL.

具体实施方式Detailed ways

定义definition

除非另外定义，否则本文所使用的全部技术术语和科学术语具有与本公开所属领域的普通技术人员通常所理解的相同意义。尽管与本文所描述的那些类似或等同的任何方法、材料、组合物、试剂、细胞可以用于实践或测试本公开的主题，但描述了优选的方法和材料。本说明书中引用的所有出版物和参考文献，包含但不限于专利和专利申请，均通过引用以其全文并入本文，如同每个单独的出版物或参考文献都具体并单独地表明为如完全阐述地通过引用并入本文。本申请要求优先权的任何专利申请也以上文对于出版物和参考文献描述的方式通过引用以其全文并入本文。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods, materials, compositions, reagents, cells similar or equivalent to those described herein can be used in the practice or testing of the disclosed subject matter, the preferred methods and materials are described. All publications and references, including but not limited to patents and patent applications, cited in this specification are hereby incorporated by reference in their entirety as if each individual publication or reference were specifically and individually indicated as if fully It is expressly incorporated herein by reference. Any patent application to which this application claims priority is also incorporated by reference in its entirety in the manner described above for publications and references.

除非特别相反地指出，否则本公开的实践将采用本领域范围内的病毒学、免疫学、微生物学、分子生物学和重组DNA技术的常规方法，其中许多方法在下文中出于说明的目的而描述。此类技术在文献中得到充分解释。参见例如，《最新蛋白质科学实验指南、最新分子生物学实验指南或最新免疫学实验指南(Current Protocols in Protein Science,Current Protocols in Molecular Biology or Current Protocols in Immunology)》，John Wiley和Sons，纽约(N.Y.)，(2009)；Ausubel等人，《精编分子生物学实验指南(ShortProtocols in Molecular Biology)》，第3版，Wiley和Sons，1995；Sambrook和Russell，《分子克隆：实验手册(Molecular Cloning:A Laboratory Manual)》(第3版，2001)；Maniatis等人，《分子克隆：实验手册》(1982)；《DNA克隆：一种实用方法(DNA Cloning:A PracticalApproach)》，第I和II卷(D.Glover编辑)；《寡核苷酸合成(Oligonucleotide Synthesis)》(N.Gait编辑，1984)；《核酸杂交(Nucleic Acid Hybridization)》(B.Hames和S.Higgins编辑，1985)；《转录和转译(Transcription and Translation)》(B.Hames和S.Higgins编辑，1984)；《动物细胞培养(Animal Cell Culture)》(R.Freshney编辑，1986)；Perbal，《分子克隆实用指南(A Practical Guide to Molecular Cloning)》(1984)以及其它相似参考文献。Unless specifically indicated to the contrary, the practice of the present disclosure will employ conventional methods of virology, immunology, microbiology, molecular biology, and recombinant DNA technology within the art, many of which are described hereinafter for purposes of illustration . Such techniques are fully explained in the literature. See, eg, Current Protocols in Protein Science, Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley and Sons, New York (N.Y. ), (2009); Ausubel et al., Short Protocols in Molecular Biology, 3rd edition, Wiley and Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual: A Laboratory Manual (3rd Edition, 2001); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, Vols I and II (Edited by D. Glover); "Oligonucleotide Synthesis" (Edited by N. Gait, 1984); "Nucleic Acid Hybridization" (Edited by B. Hames and S. Higgins, 1985); " Transcription and Translation (eds. B. Hames and S. Higgins, 1984); Animal Cell Culture (ed. R. Freshney, 1986); Perbal, A Practical Guide to Molecular Cloning (A Practical Guide to Molecular Cloning)" (1984) and other similar references.

标准技术可以用于重组DNA、寡核苷酸合成以及组织培养和转化(例如，电穿孔、脂质转染)。酶促反应和纯化技术可以根据制造商的说明书进行，或者如本领域通常实现的或如本文所述的进行。这些和相关的技术和程序通常可以根据本领域熟知的常规方法进行，并且如在本说明书通篇引用和讨论的各种一般和更具体的参考文献中所描述的。除非提供具体的定义，否则与本文所述的分子生物学、分析化学、合成有机化学以及药物和药物化学结合使用的命名和实验室程序和技术是本领域公知和常用的那些。标准技术可以用于重组技术、分子生物学、微生物学、化学合成、化学分析、药物制备、配制和递送以及患者的治疗。Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (eg, electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications, or as commonly accomplished in the art or as described herein. These and related techniques and procedures can generally be performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. Unless specific definitions are provided, the nomenclature and laboratory procedures and techniques used in connection with molecular biology, analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are those well known and commonly used in the art. Standard techniques can be used in recombinant techniques, molecular biology, microbiology, chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients.

出于本公开的目的，以下术语定义如下。For the purposes of this disclosure, the following terms are defined as follows.

本文使用冠词“一个(a)”和“一种(an)”来指一个或多于一个(即，至少一个)所述冠词的语法宾语。通过举例，“元件”意指一个元件或多于一个元件。The articles "a" and "an" are used herein to refer to one or more than one (ie, at least one) of the grammatical objects of the article. By way of example, "element" means one element or more than one element.

“约”是指量、水平、数值、数量、频率、百分比、尺寸、大小、总量、重量或长度相对于参考量、水平、数值、数量、频率、百分比、尺寸、大小、总量、重量或长度变化多达30％、25％、20％、15％、10％、9％、8％、7％、6％、5％、4％、3％、2％或1％。"About" means an amount, level, value, amount, frequency, percentage, size, size, total amount, weight or length relative to a reference amount, level, value, amount, frequency, percentage, size, size, total amount, weight Or the length varies by up to 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%.

“拮抗剂”是指干扰或以其它方式降低另一种药剂或分子的生理作用的生物结构或化学药剂。在一些情况下，拮抗剂特异性结合其它药剂或分子。包括全部和部分拮抗剂。"Antagonist" refers to a biological structure or chemical agent that interferes with or otherwise reduces the physiological effect of another agent or molecule. In some instances, the antagonist specifically binds other agents or molecules. Full and partial antagonists are included.

“激动剂”是指增加或增强另一种药剂或分子的生理作用的生物结构或化学药剂。在一些情况下，激动剂特异性结合其它药剂或分子。包括全部和部分激动剂。"Agonist" refers to a biological structure or chemical agent that increases or enhances the physiological effect of another agent or molecule. In some instances, the agonist specifically binds other agents or molecules. Includes full and partial agonists.

如本文所使用的，术语“氨基酸”旨在表示天然存在的和非天然存在的氨基酸以及氨基酸类似物和模拟物。例如，天然存在的氨基酸包含在蛋白质生物合成期间使用的20(L)-氨基酸以及其它如4-羟基脯氨酸、羟赖氨酸、锁链素、异锁链素、同型半胱氨酸、瓜氨酸和鸟氨酸。非天然存在的氨基酸包含本领域技术人员已知的例如(D)-氨基酸、正亮氨酸、正缬氨酸、对氟苯丙氨酸、乙硫氨酸等。氨基酸类似物包含天然和非天然存在的氨基酸的修饰形式。此类修饰可以包含例如氨基酸上的化学基团和部分的取代或置换，或通过氨基酸的衍生化。氨基酸模拟物包含例如有机结构，其表现出功能相似的性质，如参考氨基酸的电荷和电荷间隔特征。例如，模拟精氨酸(Arg或R)的有机结构将具有位于相似分子空间中且具有与天然存在的Arg氨基酸的侧链的e-氨基相同的迁移度的正电荷部分。模拟物还包含受约束的结构，以便维持氨基酸或氨基酸官能团的最佳间隔和电荷相互作用。本领域技术人员已知或可以确定哪些结构构成功能等同的氨基酸类似物和氨基酸模拟物。As used herein, the term "amino acid" is intended to refer to naturally occurring and non-naturally occurring amino acids as well as amino acid analogs and mimetics. For example, naturally occurring amino acids include the 20(L)-amino acids used during protein biosynthesis as well as others such as 4-hydroxyproline, hydroxylysine, cathelicidin, isocyclin, homocysteine, citrulline acid and ornithine. Non-naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine, and the like known to those of skill in the art. Amino acid analogs include modified forms of naturally and non-naturally occurring amino acids. Such modifications may include, for example, substitution or substitution of chemical groups and moieties on amino acids, or by derivatization of amino acids. Amino acid mimetics comprise, for example, organic structures that exhibit functionally similar properties, such as the charge and charge spacing characteristics of a reference amino acid. For example, an organic structure that mimics arginine (Arg or R) will have a positively charged moiety located in a similar molecular space and having the same mobility as the e-amino group of the side chain of a naturally occurring Arg amino acid. Mimics also contain constrained structures in order to maintain optimal spacing and charge interactions of amino acids or amino acid functional groups. Those of skill in the art know or can determine which structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

“生物相容的”是指通常对生物功能无害并且不会导致包含过敏原和疾病状态在内的任何程度的不可接受毒性的材料或化合物。"Biocompatible" refers to a material or compound that is generally innocuous to biological function and does not cause any degree of unacceptable toxicity, including allergens and disease states.

“编码序列”是指有助于基因的多肽产物的编码的任何核酸序列。相反，术语“非编码序列”是指不直接有助于基因的多肽产物的编码的任何核酸序列。"Coding sequence" refers to any nucleic acid sequence that facilitates the encoding of the polypeptide product of a gene. In contrast, the term "non-coding sequence" refers to any nucleic acid sequence that does not directly contribute to the encoding of the polypeptide product of a gene.

贯穿本公开内容，除非上下文另有要求，否则词语“包括(comprise)”、“包括了(comprises)”和“包括着(comprising)”将被理解为暗示包括所陈述的步骤或要素或一组步骤或要素但不排除任何其它步骤或要素或一组步骤或要素。Throughout this disclosure, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" will be understood to imply the inclusion of a stated step or element or group of step or element but does not exclude any other step or element or group of steps or elements.

“由……组成”意味着包含并限于短语“由……组成”之后的任何内容。因此，短语“由……组成”指示所列出的要素是必需的或强制性的并且可以不存在其它要素。“基本上由……组成”意指包含所述短语之后所列出的任何要素，并且限于不干扰或促进本公开中针对所列要素指定的活动或行为的其它要素。因此，短语“基本上由……组成”表示所列要素是必需的或强制性的，但是其它要素是任选的并且可以存在或不存在，这取决于其是否实质上影响所列要素的活性或作用。"Consisting of" is meant to include and be limited to anything following the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed element is required or mandatory and that other elements may be absent. "Consisting essentially of" is meant to include any of the elements listed following the phrase, and is limited to other elements that do not interfere with or facilitate the activities or behaviors specified in this disclosure for the listed elements. Thus, the phrase "consisting essentially of" means that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending on whether they substantially affect the activity of the listed elements or effect.

如本文所述，术语“缀合物”是指由至少两种单独的多肽(例如，第一多肽和第二多肽)共价或非共价附接或连接而形成的实体。缀合多肽的一个实例是“融合蛋白”或“融合多肽”，即通过连接两个或更多个编码序列而产生的多肽，所述编码序列最初编码单独的多肽；结合的编码序列的翻译产生单一的融合多肽，其通常具有衍生自每个单独的多肽的功能性质。As used herein, the term "conjugate" refers to an entity formed by the covalent or non-covalent attachment or linkage of at least two separate polypeptides (eg, a first polypeptide and a second polypeptide). An example of a conjugated polypeptide is a "fusion protein" or "fusion polypeptide," ie, a polypeptide produced by linking two or more coding sequences that originally encoded separate polypeptides; translation of the combined coding sequences produces A single fusion polypeptide, which generally has functional properties derived from each individual polypeptide.

术语“不含内毒素”或“基本上不含内毒素”通常涉及至多含有痕量(例如，对受试者没有临床不利生理作用的量)内毒素以及优选地无法检测到的量的内毒素的组合物、溶剂和/或血管。内毒素是与某些微生物，如细菌(典型地革兰氏阴性细菌)相关的毒素，尽管可能在如单核细胞增生李斯特菌等革兰氏阳性细菌中发现内毒素。最普遍的内毒素是在各种革兰氏阴性细菌的外膜中发现的并且表示这些细菌在引起疾病的能力方面的中心致病特征的脂多糖(LPS)或脂低聚糖(LOS)。人体中的少量内毒素可能产生发热、血压降低、炎症和凝血激活以及其它不良生理影响。The terms "free of endotoxin" or "substantially free of endotoxin" generally relate to containing at most trace amounts (eg, amounts that have no clinically adverse physiological effect on the subject) endotoxins and preferably undetectable amounts of endotoxins composition, solvent and/or blood vessel. Endotoxins are toxins associated with certain microorganisms, such as bacteria (typically Gram-negative bacteria), although endotoxins may be found in Gram-positive bacteria such as Listeria monocytogenes. The most prevalent endotoxins are lipopolysaccharides (LPS) or lipo-oligosaccharides (LOS), which are found in the outer membranes of various Gram-negative bacteria and represent the central pathogenic feature of these bacteria in their ability to cause disease. Small amounts of endotoxins in humans may produce fever, lowering of blood pressure, inflammation and activation of coagulation, and other adverse physiological effects.

因此，在药物生产中，常常期望从药物产品和/或药物容器中除去大部分或全部痕量的内毒素，因为即使少量也可能对人类产生不良影响。除热原烘箱可以用于此目的，因为通常需要超过300℃的温度来分解大多数内毒素。例如，基于如注射器或小瓶等初级包装材料，250℃的玻璃温度和30分钟的保持时间的组合常常足以实现内毒素水平的3log减少。设想了除去内毒素的其它方法，例如如本文所描述的和本领域中已知的色谱法和过滤法。Thus, in pharmaceutical production, it is often desirable to remove most or all trace amounts of endotoxins from pharmaceutical products and/or pharmaceutical containers, since even small amounts may have adverse effects on humans. A depyrogenation oven can be used for this purpose, as temperatures in excess of 300°C are typically required to decompose most endotoxins. For example, based on primary packaging materials such as syringes or vials, the combination of a glass temperature of 250°C and a hold time of 30 minutes is often sufficient to achieve a 3 log reduction in endotoxin levels. Other methods of removing endotoxin are contemplated, such as chromatography and filtration as described herein and known in the art.

可以使用本领域已知的常规技术检测内毒素。例如，利用来自马蹄蟹的血液的鲎变形细胞溶解物测定法是用于检测内毒素存在的非常灵敏的测定法。在这个测试中，非常低水平的LPS就可以引起鲎裂解物的可检测到的凝血，这是由于强大的酶级联放大了这一反应。内毒素也可以通过酶联免疫吸附测定(ELISA)进行定量。为基本上不含内毒素，内毒素水平可以低于活性化合物的约0.001、0.005、0.01、0.02、0.03、0.04、0.05、0.06、0.08、0.09、0.1、0.5、1.0、1.5、2、2.5、3、4、5、6、7、8、9或10EU/mg。通常，1ng脂多糖(LPS)对应于约1-10EU。Endotoxins can be detected using conventional techniques known in the art. For example, the Limulus amoebocyte lysate assay using blood from horseshoe crabs is a very sensitive assay for detecting the presence of endotoxin. In this test, very low levels of LPS caused detectable coagulation of the Limulus lysate due to a powerful enzyme cascade that amplifies the reaction. Endotoxins can also be quantified by enzyme-linked immunosorbent assay (ELISA). To be substantially free of endotoxin, the endotoxin level may be less than about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9 or 10 EU/mg. Typically, 1 ng of lipopolysaccharide (LPS) corresponds to about 1-10 EU.

缀合物或多肽的“半衰期”可以指缀合物或多肽相对于在施用于生物体的血清或组织时的此类活性、或相对于任何其它限定的时间点丧失其药理学、生理学或其它活性的一半所花费的时间。“半衰期”也可以指缀合物或多肽的量或浓度相对于施用于生物体血清或组织时的量或浓度，或相对于任何其它限定的时间点，减少施用于生物体血清或组织中的起始量的一半所花费的时间。可以在血清和/或任何一种或多种所选组织中测量半衰期。The "half-life" of a conjugate or polypeptide may refer to the loss of pharmacological, physiological or other properties of the conjugate or polypeptide relative to such activity when administered to the serum or tissue of an organism, or relative to any other defined point in time. half the time spent active. "Half-life" may also refer to the amount or concentration of a conjugate or polypeptide relative to the amount or concentration when administered to the serum or tissue of the organism, or relative to any other defined point in time, that reduces the amount or concentration of the conjugate or polypeptide administered to the serum or tissue of the organism The time spent in half the starting amount. Half-life can be measured in serum and/or any one or more selected tissues.

术语“调节”和“改变”包含“增加”、“增强”或“刺激”，以及“降低”或“减少”，通常相对于对照具有统计学显著或生理学显著的量或程度。“增加的”、“刺激的”或“增强的”量通常是“统计上显著的”量，并且可以包含1.1、1.2、2、3、4、5、6、7、8、9、10、15、20、30或更多倍(例如，500、1000倍)(包含所有整数和其间的范围，例如，1.5、1.6、1.7、1.8等)的由非组合物(例如，无药剂)或对照组合物产生的量的增加。“降低”或“减少”量通常是“统计上显著”的量，并且可以包含1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、11％、12％、13％、14％、15％、16％、17％、18％、19％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或100％(包含所有整数和其间的范围)的非组合物(例如，无药剂)或对照组合物产生的量的减少。本文描述了比较和“统计学显著”量的实例。The terms "modulate" and "change" include "increase," "enhance," or "stimulate," as well as "decrease" or "decrease," usually by a statistically or physiologically significant amount or degree relative to a control. An "increased", "stimulated" or "enhanced" amount is usually a "statistically significant" amount and can include 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (eg, 500, 1000 times) (including all integers and ranges therebetween, eg, 1.5, 1.6, 1.7, 1.8, etc.) from a non-composition (eg, no agent) or control An increase in the amount produced by the composition. A "reduced" or "reduced" amount is usually a "statistically significant" amount and may comprise 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% , 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55 %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (including all integers and ranges therebetween) of the non-composition (eg, no agent) or control combination reduction in the amount of biomass produced. Examples of comparative and "statistically significant" quantities are described herein.

术语“多肽”、“蛋白质”和“肽”可互换使用，并且意指不限于任何特定长度的氨基酸的聚合物。术语“酶”包含多肽或蛋白质催化剂，并且就ADI而言，其可与蛋白质、多肽或肽互换使用。所述术语包含如豆蔻酰化、硫酸化、糖基化、磷酸化和信号序列的添加或缺失等修饰。术语“多肽”或“蛋白质”意指一个或多个氨基酸链，其中每个链包括通过肽键共价连接的氨基酸，并且其中所述多肽或蛋白质可以包括多个通过肽键非共价和/或共价连接且具有天然蛋白质序列的链，即，由天然存在的以及特别是非重组细胞、或基因工程或重组细胞产生的蛋白质，并且包括具有天然蛋白质的氨基酸序列的分子、或具有天然序列的一个或多个氨基酸的缺失、添加和/或取代的分子。术语“多肽”和“蛋白质”具体地包括本文所述的ADI酶/蛋白质，或具有ADI蛋白质的一个或多个氨基酸的缺失、添加和/或取代的序列。在某些实施例中，多肽是由重组细胞产生的“重组”多肽，所述重组细胞包括一个或多个重组DNA分子，所述重组DNA分子通常由在细胞中无法以其它方式找到的异源多核苷酸序列或多核苷酸序列的组合制成。The terms "polypeptide", "protein" and "peptide" are used interchangeably and are meant to refer to polymers of amino acids not limited to any particular length. The term "enzyme" includes polypeptides or protein catalysts and is used interchangeably with proteins, polypeptides or peptides with respect to ADI. The term includes modifications such as myristoylation, sulfation, glycosylation, phosphorylation and addition or deletion of signal sequences. The term "polypeptide" or "protein" means one or more chains of amino acids, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein the polypeptide or protein may comprise a plurality of non-covalent and/or multiple peptide bonds or covalently linked chains with native protein sequences, i.e. proteins produced by naturally occurring and in particular non-recombinant cells, or genetically engineered or recombinant cells, and including molecules with the amino acid sequences of native proteins, or with native sequences Molecules with deletions, additions and/or substitutions of one or more amino acids. The terms "polypeptide" and "protein" specifically include the ADI enzymes/proteins described herein, or sequences having deletions, additions and/or substitutions of one or more amino acids of an ADI protein. In certain embodiments, the polypeptide is a "recombinant" polypeptide produced by a recombinant cell that includes one or more recombinant DNA molecules, typically derived from a heterologous source not otherwise found in the cell A polynucleotide sequence or combination of polynucleotide sequences is made.

本文提及的术语“分离的”多肽或蛋白质是指受试蛋白质：(1)不含至少一些其它蛋白质，通常将使用所述至少一些其它蛋白质在自然界中发现所述受试蛋白质；(2)基本上不含来自相同来源，例如，来自相同物种的其它蛋白质；(3)由来自不同物种的细胞表达；(4)已与至少约50％的多核苷酸、脂质、碳水化合物或与其天然缔合的其它物质分离；(5)不和与“分离的蛋白质”天然缔合的蛋白质部分缔合(通过共价或非共价相互作用)；(6)和不与其天然缔合的多肽可操作地缔合(通过共价或非共价相互作用)；或(7)在自然界中不存在。这种分离的蛋白质可以由基因组DNA、cDNA、mRNA或其它RNA编码，或者可以具有合成来源，或其任何组合。在某些实施例中，分离的蛋白质基本上不含在其天然环境中会发现的且会干扰其使用(治疗、诊断、预防、研究或其它)的蛋白质或多肽或其它污染物。As used herein, the term "isolated" polypeptide or protein refers to a test protein: (1) free of at least some other protein with which the test protein would normally be found in nature; (2) be substantially free of other proteins from the same source, eg, from the same species; (3) expressed by cells from a different species; (4) bound to at least about 50% of polynucleotides, lipids, carbohydrates, or native Other substances that are associated are separated; (5) are not associated with the protein moiety with which the "isolated protein" is naturally associated (through covalent or non-covalent interactions); (6) the polypeptide may not be naturally associated with it is operatively associated (through covalent or non-covalent interactions); or (7) does not occur in nature. Such isolated proteins may be encoded by genomic DNA, cDNA, mRNA, or other RNA, or may be of synthetic origin, or any combination thereof. In certain embodiments, the isolated protein is substantially free of proteins or polypeptides or other contaminants that would be found in its natural environment and that would interfere with its use (therapeutic, diagnostic, prophylactic, research, or otherwise).

在某些实施例中，组合物中任何给定药剂(例如，缀合物)的“纯度”可以被具体定义。例如，某些组合物可以包括纯度至少为80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％(包含所有小数和其间的范围)的缀合物，例如且绝不限制，通过高效液相色谱法(HPLC)测量的，高效液相色谱法是在生物化学和分析化学中常用的用来分离、鉴定和定量化合物的公知形式柱色谱法。在一些情况下，组合物的纯度以聚集度为特征。例如，缀合物(例如，融合蛋白)的聚集度可以通过尺寸排阻色谱(SEC)来确定，所述尺寸排阻色谱根据尺寸来分离颗粒。所述SEC是用于确定纯化蛋白质的三级结构和四级结构的普遍接受的方法。SEC主要用于分析大分子，如蛋白质或聚合物。SEC是通过将这些较小的分子捕获在颗粒的孔中来工作的。较大的分子只是通过孔，因为它们太大而不能进入孔。因此，较大分子比较小分子更快地流过柱，即分子越小，保留时间越长。某些组合物也基本上不含聚集物(大于约95％，通过SEC HPLC呈现为单峰)。某些实施例不含具有大于约96％、约97％、约98％或约99％的聚集物，通过SEC HPLC呈现为单峰。In certain embodiments, the "purity" of any given agent (eg, conjugate) in a composition can be specifically defined. For example, certain compositions may include a purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% ( Conjugates including all decimals and ranges therebetween, for example and in no way limiting, as measured by high performance liquid chromatography (HPLC), a method commonly used in biochemistry and analytical chemistry to separate, A well-known form of column chromatography for the identification and quantification of compounds. In some cases, the purity of the composition is characterized by the degree of aggregation. For example, the degree of aggregation of a conjugate (eg, a fusion protein) can be determined by size exclusion chromatography (SEC), which separates particles according to size. The SEC is a generally accepted method for determining the tertiary and quaternary structures of purified proteins. SEC is mainly used to analyze macromolecules such as proteins or polymers. SEC works by trapping these smaller molecules in the pores of the particles. Larger molecules simply pass through the pores because they are too large to enter the pores. Thus, larger molecules flow through the column faster than smaller molecules, ie, the smaller the molecule, the longer the retention time. Certain compositions were also substantially free of aggregates (greater than about 95%, as a single peak by SEC HPLC). Certain embodiments are free of aggregates having greater than about 96%, about 97%, about 98%, or about 99% as a single peak by SEC HPLC.

术语“参考序列”通常是指与另一序列正在进行比较的核酸编码序列或氨基酸序列。本文所述的所有多肽和多核苷酸序列都作为参考序列而包含，包含通过名称描述的参考序列和在表和序列表中描述的参考序列。The term "reference sequence" generally refers to a nucleic acid coding sequence or amino acid sequence to which another sequence is being compared. All polypeptide and polynucleotide sequences described herein are included as reference sequences, including reference sequences described by name and reference sequences described in the Tables and Sequence Listing.

如本文所使用的术语“序列一致性”或例如，包括“与之50％一致的序列”是指在比较窗口内，在逐核苷酸的基础上或在逐氨基酸的基础上序列一致的程度。因此，“序列一致性百分比”可以通过以下方式计算：在所述比较窗口内比较两个经最佳比对的序列，测定在这两个序列中出现一致的核酸碱基(例如，A、T、C、G、I)或一致的氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys以及Met)的位置的数目，从而得到匹配的位置的数目，将匹配的位置的数目除以所述比较窗口中的位置的总数目(即，窗口大小)，并且将所述结果乘以100，得到序列一致性百分比。用于比对比较窗口的序列的最佳比对可以通过算法(美国威斯康星州麦迪逊科学大道575号遗传学电脑集团的威斯康星州遗传学软件包7.0版本中的GAP、BESTFIT、FASTA和TFASTA)的计算机化实施方式，或者通过检验各种所选方法中的任何方法产生的最佳比对(即，导致比较窗口内的最高百分比同源性)而进行。还可以参考例如由Altschul等人，《核酸研究(NuclAcids Res.)》25:3389,1997所公开的BLAST程序家族。The term "sequence identity" or, for example, including "sequences that are 50% identical to" as used herein refers to the degree of sequence identity on a nucleotide-by-nucleotide basis or on an amino acid-by-amino acid basis within a window of comparison . Thus, "percent sequence identity" can be calculated by comparing two optimally aligned sequences within the comparison window and determining the nucleic acid bases (eg, A, T) that appear identical in the two sequences , C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) to obtain the number of matched positions, divide the number of matched positions by the total number of positions in the comparison window (ie, the window size), and multiply the result by With 100, the percent sequence identity is obtained. Optimal alignment of sequences for alignment comparison windows can be achieved by algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Package Version 7.0, Genetics Computer Group, 575 Science Avenue, Madison, Wisconsin, USA). computerized embodiments, or by examining any of a variety of selected methods that yields the best alignment (ie, that results in the highest percent homology within the comparison window). Reference can also be made to the family of BLAST programs disclosed, for example, by Altschul et al., Nucl Acids Res. 25:3389, 1997.

术语“溶解度”是指本文所述药剂(例如，缀合物)溶解在液体溶剂中并形成均匀溶液的性质。溶解度通常表示为浓度，是每单位体积溶剂的溶质质量(每千克溶剂的溶质克数、g/dL(100mL)、mg/mL等)、摩尔浓度、质量摩尔浓度、摩尔分数或其它类似的浓度描述。可溶解每种单位量溶剂的溶质的最大均衡量是所述溶质在包括温度、压力、pH和溶剂的性质的特定条件下在所述溶剂中的溶解度。在某些实施例中，在生理pH或其它pH，例如，pH 5.0、pH 6.0、pH 7.0、pH 7.4、pH 7.6、pH 7.8、或pH 8.0(例如，约pH 5-8)下测量溶解度。在某些实施例中，在水或如PBS或NaCl(有或无NaP)的生理缓冲液中测量溶解度。在具体实施例中，在相对较低的pH(例如，pH 6.0)和相对较高的盐(例如，500mM的NaCl和10mM的NaP)下测量溶解度。在某些实施例中，在如血液或血清的生物流体(溶剂)中测量溶解度。在某些实施例中，温度可以约为室温(例如，约20℃、21℃、22℃、23℃、24℃、25℃)或约为体温(37℃)。在某些实施例中，药剂在室温或37℃下具有至少约0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、25、30、40、50、60、70、80、90或100mg/mL的溶解度。The term "solubility" refers to the property of an agent (eg, a conjugate) described herein to dissolve in a liquid solvent and form a homogeneous solution. Solubility is usually expressed as concentration and is the mass of solute per unit volume of solvent (grams of solute per kilogram of solvent, g/dL (100 mL), mg/mL, etc.), molarity, molality, mole fraction, or other similar concentrations describe. The maximum equilibrium amount of solute that can dissolve each unit amount of solvent is the solubility of the solute in the solvent under specified conditions including temperature, pressure, pH, and properties of the solvent. In certain embodiments, solubility is measured at physiological pH or other pH, eg, pH 5.0, pH 6.0, pH 7.0, pH 7.4, pH 7.6, pH 7.8, or pH 8.0 (eg, about pH 5-8). In certain embodiments, solubility is measured in water or a physiological buffer such as PBS or NaCl (with or without NaP). In particular embodiments, solubility is measured at relatively low pH (eg, pH 6.0) and relatively high salt (eg, 500 mM NaCl and 10 mM NaP). In certain embodiments, solubility is measured in biological fluids (solvents) such as blood or serum. In certain embodiments, the temperature may be about room temperature (eg, about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C) or about body temperature (37°C). In certain embodiments, the agent has at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mg/mL solubility.

“受试者”或“有需要的受试者”或“患者”或“有需要的患者”包含哺乳动物受试者如受试的人。"Subject" or "subject in need" or "patient" or "patient in need" includes mammalian subjects such as a human subject.

“基本上”或“实质上”意指几乎完全或完全，例如，一些给定量的95％、96％、97％、98％、99％或更高。"Substantially" or "substantially" means almost completely or completely, eg, 95%, 96%, 97%, 98%, 99% or more of some given amount.

“统计上显著”意味着结果不太可能偶然发生。统计显著性可以通过本领域已知的任何方法来确定。常用的显著性度量包含p值，如果零假设为真，则p值是发现事件发生的频率或概率。如果获得的p值小于显著性水平，则零假设被否定。在简单的情况下，显著性水平限定在0.05或更小的p值。"Statistically significant" means that the result is unlikely to occur by chance. Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which is the frequency or probability of finding an event if the null hypothesis is true. If the obtained p-value is less than the significance level, the null hypothesis is rejected. In the simple case, the significance level was limited to a p-value of 0.05 or less.

“治疗反应”是指基于一种或多种治疗剂(例如，缀合物)的施用改善症状(无论是否持续)。"Therapeutic response" refers to amelioration of symptoms (whether sustained or not) upon administration of one or more therapeutic agents (eg, conjugates).

如本文所使用的，受试者(例如哺乳动物，如人)或细胞的“治疗”是用于试图改变个体或细胞的自然进程的任何类型的干预。治疗包含但不限于施用药物组合物，并且可以预防性地或在病理事件开始后或与病原体接触后进行。还包含“预防性”治疗，其可以针对于降低所治疗的疾病或病症的进展速率、延迟所述疾病或病症的发作、或降低其发病的严重程度。“治疗”或“预防”不一定表示完全根除、治愈或预防疾病或病症或其相关症状。As used herein, "treatment" of a subject (eg, a mammal, such as a human) or a cell is any type of intervention used to attempt to alter the natural course of the individual or cell. Treatment includes, but is not limited to, administration of a pharmaceutical composition, and can be performed prophylactically or after the onset of a pathological event or exposure to a pathogen. Also included are "prophylactic" treatments, which may be directed to reducing the rate of progression, delaying the onset, or reducing the severity of the onset of the disease or disorder being treated. "Treatment" or "prevention" does not necessarily mean complete eradication, cure or prevention of a disease or disorder or its associated symptoms.

术语“野生型”是指在群体中最常观察到的基因或基因产物(例如，多肽)，并且因此被任意地称为基因的“正常”或“野生型”形式。The term "wild-type" refers to a gene or gene product (eg, a polypeptide) that is most commonly observed in a population, and is thus arbitrarily referred to as the "normal" or "wild-type" form of the gene.

除非另外明确地说明，否则本说明书中的每个实施例在进行必要的修改后适用于所有其它实施例。Each embodiment in this specification applies mutatis mutandis to all other embodiments unless expressly stated otherwise.

缀合物conjugate

本公开的实施例部分涉及以下意外发现：精氨酸脱亚胺酶(ADI)与肿瘤坏死因子(TNF)超家族配体(例如，TRAIL)的缀合(例如，融合)相对于单独的一种或两种组分改善了缀合物的药代动力学和/或生物活性，并且在许多情况下协同作用亦如此。还涉及以下发现：六聚体或同源六聚体多肽与三聚体或同源三聚体多肽的缀合相对于单独的一种或两种组分改善了缀合物的药代动力学和/或生物活性。还涉及以下发现：第一三聚体多肽与第二三聚体多肽(与第一三聚体多肽不同)的缀合相对于单独的一种或两种组分改善了缀合物的药代动力学和/或生物活性。在一些情况下，缀合物的每种组分增强(例如，协同增强)其它组分的药代动力学和/或生物活性。The Examples section of the present disclosure relates to the unexpected discovery that conjugation (eg, fusion) of arginine deiminase (ADI) to a ligand of the tumor necrosis factor (TNF) superfamily (eg, TRAIL) relative to a single One or both components improve the pharmacokinetics and/or biological activity of the conjugate, and in many cases do so synergistically. Also related to the discovery that conjugation of a hexameric or homohexameric polypeptide to a trimeric or homotrimeric polypeptide improves the pharmacokinetics of the conjugate relative to one or both components alone and/or biological activity. Also related to the discovery that conjugation of a first trimeric polypeptide to a second trimeric polypeptide (different from the first trimeric polypeptide) improves the pharmacokinetics of the conjugate relative to one or both components alone kinetics and/or biological activity. In some cases, each component of the conjugate enhances (eg, synergistically enhances) the pharmacokinetics and/or biological activity of the other components.

因此，某些实施例涉及缀合物，其包括与TNF超家族配体(例如，TRAIL)共价连接的精氨酸脱亚胺酶ADI，其中每一个TNF超家族配体在本文作了更为详细的描述。在一些实施例中，ADI缀合到TNF超家族配体的N端。在一些实施例中，ADI缀合到TNF超家族配体的C端。Accordingly, certain embodiments relate to conjugates comprising arginine deiminase ADI covalently linked to a TNF superfamily ligand (eg, TRAIL), wherein each TNF superfamily ligand is further described herein for a detailed description. In some embodiments, ADI is conjugated to the N-terminus of a TNF superfamily ligand. In some embodiments, ADI is conjugated to the C-terminus of a TNF superfamily ligand.

还包含缀合物，其包括(a)共价连接到三聚体多肽的六聚体多肽，或(b)共价连接到第二三聚体多肽的第一三聚体多肽，所述第二三聚体多肽与所述第一三聚体多肽不同。在一些情况下，六聚体多肽是同源六聚体多肽。(a)的六聚体或同源六聚体多肽的实例包含例如某些ADI，如来自鸽支原体、惰性支原体、鸡支原体和火鸡支原体的天然ADI(例如，分别为SEQ ID NO:9、37、38、50)，来自表A1的嵌合ADI(例如，SEQID NO:57-68)，以及脂联素或其胶原样结构域，这些在本文作了更为详细的描述。脂联素是244个氨基酸的蛋白质，其由氨基末端信号肽、N端处胶原样结构域和C端处球状结构域构成。脂联素自缔合成更大的结构，例如，脂联素分子通过胶原样结构域结合在一起形成同源三聚体，并且在一些情况下三聚体继续自缔合并形成六聚体。Also included are conjugates comprising (a) a hexameric polypeptide covalently linked to a trimeric polypeptide, or (b) a first trimeric polypeptide covalently linked to a second trimeric polypeptide, the The ditrimeric polypeptide is different from the first trimeric polypeptide. In some instances, the hexameric polypeptide is a homohexameric polypeptide. Examples of hexameric or homohexameric polypeptides of (a) include, for example, certain ADIs such as native ADIs from M. pigeon, M. inert, M. gallinarum, and M. turkeyi (eg, SEQ ID NO: 9, 37, 38, 50), chimeric ADIs from Table Al (eg, SEQ ID NOs: 57-68), and adiponectin or its collagen-like domains, which are described in more detail herein. Adiponectin is a 244 amino acid protein composed of an amino-terminal signal peptide, a collagen-like domain at the N-terminus, and a globular domain at the C-terminus. Adiponectin self-associates into larger structures, eg, adiponectin molecules are held together by collagen-like domains to form homotrimers, and in some cases trimers continue to self-associate and form hexamers.

在一些情况下，三聚体多肽是同源三聚体多肽。(b)的第一三聚体或同源三聚体多肽的实例包含脂联素或其胶原样结构域、T4次要纤维蛋白或其三聚结构域(折叠)、前胶原的C-前肽、表面活性蛋白A(SP-A)和甘露糖结合蛋白A(MBP-A)。如上所述，在一些情况下，脂联素或其胶原样结构域自缔合成三聚体。噬菌体T4次要纤维蛋白是三链、平行、分段的α螺旋卷曲蛋白。T4次要纤维蛋白的C端球状结构域(折叠)对于正确的三聚化和蛋白质折叠是必不可少的，然而在没有次要纤维蛋白的卷曲螺旋部分的情况下，折叠能够三聚化(参见Letarov等人，《生物化学(莫斯科)(Biochemistry(Mosc)》，64(7):817-23,1999)。纤维状前胶原的C-前肽通过控制前胶原分子的细胞内组装和胶原纤维的细胞外组装，在组织生长和修复中起关键作用，并且负责在各种前胶原之间选择性形成同源三聚体和某些异源三聚体(参见，例如，Bourhis等人，《自然结构和分子生物学(Nat Struct Mol Biol.)》19(10):1031-1036,2012)。表面活性蛋白A(SP-A)是与肺表面活性剂相关的四种蛋白质之一，其与肺泡磷脂膜以高亲和力结合，将蛋白质定位在抵抗吸入性病原体的第一道防线上。SP-A既表现出钙依赖性碳水化合物结合(胶原凝集素家族的特性)，又表现出与脂膜组分的特异性相互作用。SP-A的碳水化合物识别结构域(CRD)与颈部结构域形成三聚体结构(参见例如，《生物化学杂志(J.Biol.Chem.)》278(44):43254-60,2003)。甘露糖结合蛋白(MBP)是血清中发现的C型(Ca(2+)-依赖性)动物凝集素。这些MBP识别病原菌和真菌特有的细胞表面寡糖结构，并触发这些生物体的中和。MBP的碳水化合物识别结构域(CRD)和将羧基末端CRD与完整分子的胶原样部分连接的颈部结构域形成三聚体结构(参见例如，Weis和Drickamer，《结构(Structure.)》2(12):1227-40,1994)。因此，任何前述三聚体多肽或其三聚体片段/结构域可用作(b)的第一三聚体多肽。In some instances, the trimeric polypeptide is a homotrimeric polypeptide. Examples of the first trimer or homotrimeric polypeptide of (b) include adiponectin or its collagen-like domain, T4 minor fibrin or its trimerization domain (fold), C-procollagen Peptides, Surfactant Protein A (SP-A) and Mannose Binding Protein A (MBP-A). As noted above, in some instances, adiponectin or its collagen-like domains self-associate into trimers. Phage T4 minor fibrin is a triple-stranded, parallel, segmented alpha-helical coiled protein. The C-terminal globular domain (fold) of the T4 minor fibrin is essential for proper trimerization and protein folding, however in the absence of the coiled-coil portion of the minor fibrin, the fold is capable of trimerization ( See Letarov et al., Biochemistry (Mosc), 64(7):817-23, 1999. The C-propeptide of fibrillar procollagen works by controlling intracellular assembly of procollagen molecules and collagen Extracellular assembly of fibers, which plays a key role in tissue growth and repair, and is responsible for the selective formation of homotrimers and some heterotrimers among various procollagens (see, e.g., Bourhis et al., "Nat Struct Mol Biol." 19(10):1031-1036, 2012). Surfactant protein A (SP-A) is one of four proteins associated with pulmonary surfactant, It binds with high affinity to alveolar phospholipid membranes, positioning the protein in the first line of defense against inhaled pathogens. SP-A exhibits both calcium-dependent carbohydrate binding (property of the agglutinin family) and Specific interactions of lipid membrane components. The carbohydrate recognition domain (CRD) of SP-A forms a trimeric structure with the neck domain (see, eg, J. Biol. Chem. 278 (44):43254-60, 2003). Mannose-binding proteins (MBPs) are C-type (Ca(2+)-dependent) animal lectins found in serum. These MBPs recognize cell surface oligosaccharides specific to pathogenic bacteria and fungi carbohydrate structures, and trigger neutralization in these organisms. The carbohydrate recognition domain (CRD) of MBP and the neck domain linking the carboxy-terminal CRD to the collagen-like portion of the intact molecule form a trimeric structure (see e.g., Weis and Drickamer, "Structure." 2(12):1227-40, 1994). Thus, any of the foregoing trimeric polypeptides or trimeric fragments/domains thereof can be used as the first trimer of (b) body polypeptide.

在一些实施例中，(a)的三聚体或同源三聚体多肽，或(b)的第二三聚体或同源三聚体多肽是TNF超家族配体或受体，其在本文作了更为详细的描述。In some embodiments, the trimeric or homotrimeric polypeptide of (a), or the second trimeric or homotrimeric polypeptide of (b) is a TNF superfamily ligand or receptor that is in This article provides a more detailed description.

在一些实施例中，对于(a)，六聚体多肽共价连接到三聚体多肽的N端，或者对于(b)，第一三聚体多肽共价连接到第二三聚体多肽的N端。在一些实施例中，对于(a)，六聚体多肽共价连接到三聚体多肽的C端，或者对于(b)，第一三聚体多肽共价连接到第二三聚体多肽的C端。In some embodiments, for (a) the hexameric polypeptide is covalently linked to the N-terminus of the trimeric polypeptide, or for (b) the first trimeric polypeptide is covalently linked to the second trimeric polypeptide N-terminal. In some embodiments, for (a), the hexameric polypeptide is covalently linked to the C-terminus of the trimeric polypeptide, or for (b), the first trimeric polypeptide is covalently linked to the second trimeric polypeptide C-terminal.

在一些情况下，缀合物是融合蛋白，例如，缀合物的两个组分之间的共价键完全由肽键构成。在一些情况下，缀合物是非融合蛋白，例如，其中缀合物组分之间的共价键包括至少一个非肽键，或者其中共价键在缀合物的每个多肽已经单独产生(例如，重组产生)并任选地纯化后发生化学反应。In some cases, the conjugate is a fusion protein, eg, the covalent bond between the two components of the conjugate consists entirely of peptide bonds. In some cases, the conjugate is a non-fusion protein, for example, wherein the covalent bond between the components of the conjugate includes at least one non-peptide bond, or wherein the covalent bond has been created separately in each polypeptide of the conjugate ( For example, recombinant production) and optionally purification followed by chemical reaction.

在一些实施例中，缀合物包括缀合物的每个组分之间的连接子(例如，生理稳定的连接子)。连接子的一般实例包含肽连接子(例如，柔性连接子和刚性连接子)和非肽连接子。本文对示例性连接子作了更为详细地描述。In some embodiments, the conjugate includes a linker (eg, a physiologically stable linker) between each component of the conjugate. General examples of linkers include peptide linkers (eg, flexible linkers and rigid linkers) and non-peptide linkers. Exemplary linkers are described in more detail herein.

在一些情况下，如上所述，缀合物的至少一种组分改善了缀合物的另一种组分的一种或多种性质，并且在一些情况下，缀合物协同作用亦如此。在一些情况下，每种组分改善了缀合物的另一种组分的一种或多种性质。在一些情况下，相对于单独的一种或两种组分，缀合物具有一种或多种改进的性质。示例性性质包含物理和/或药代动力学性质，如蛋白质稳定性、溶解度、血清半衰期、生物利用度、暴露度和清除率。还包含生物性质或活性。In some cases, as described above, at least one component of the conjugate improves one or more properties of another component of the conjugate, and in some cases, the conjugate acts synergistically . In some cases, each component improves one or more properties of the other component of the conjugate. In some cases, the conjugate has one or more improved properties relative to one or both components alone. Exemplary properties include physical and/or pharmacokinetic properties, such as protein stability, solubility, serum half-life, bioavailability, exposure, and clearance. Also includes biological properties or activities.

在一些情况下，相对于单独的一种或两种组分，缀合物的生物活性增加。在一些情况下，相对于单独的每种组分，缀合物对生物活性具有“加和”效应。“加和性”是指增加的缀合物活性，其约等于单独的每种组分组合的加和活性。在一些情况下，相对于单独的每种组分，缀合物对生物活性具有“协同”作用。“协同作用”或“协合作用”是指增加的缀合物活性，其大于单独的每种组分的组合的加和活性。在一些情况下，缀合物的一种组分“增强”另一种组分的生物活性(例如，在一些情况下，ADI增强TRAIL的活性)。“增强”是指在只有一种组分单独是活性的或显著活性的情况下缀合物活性增加。在一些情况下，缀合物的组分之间存在“相互作用(coalism)”，其是指在两种组分本身都不具有活性的情况下的缀合物活性。In some cases, the biological activity of the conjugate is increased relative to one or both components alone. In some cases, the conjugate has an "additive" effect on biological activity relative to each component alone. "Additive" refers to an increased conjugate activity that is approximately equal to the additive activity of each combination of components individually. In some cases, the conjugate has a "synergistic" effect on biological activity relative to each component alone. "Synergy" or "synergy" refers to an increased conjugate activity that is greater than the additive activity of the combination of each component individually. In some instances, one component of the conjugate "enhances" the biological activity of another component (eg, in some instances, ADI enhances the activity of TRAIL). "Enhancing" refers to an increase in conjugate activity where only one component alone is active or significantly active. In some cases, there is "coalism" between the components of the conjugate, which refers to the activity of the conjugate where neither component is active on its own.

在具体实施例中，缀合物包括共价连接到TNF超家族成员配体(例如，TRAIL、TNF-α、FasL)的ADI，并且相对于单独的ADI和/或单独的TNF超家族配体，缀合物具有改善的药代动力学、物理和/或生物学性质。如上所述，示例性药代动力学和物理性质包含增加的稳定性、增加的血清半衰期、增加的生物利用度、增加的暴露度和降低的清除率。在一些情况下，相对于单独的ADI和/或单独的TNF超家族配体，缀合物具有增加的稳定性和/或血清半衰期。在一些情况下，相对于单独的ADI和/或单独的TNF超家族配体，缀合物的稳定性和/或血清半衰期增加了约或至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、200％、300％、400％、500％、600％、700％、800％、900％或1000％或更多。In specific embodiments, the conjugate comprises ADI covalently linked to a TNF superfamily member ligand (eg, TRAIL, TNF-α, FasL), and is relative to ADI alone and/or TNF superfamily ligand alone , the conjugate has improved pharmacokinetic, physical and/or biological properties. As mentioned above, exemplary pharmacokinetic and physical properties include increased stability, increased serum half-life, increased bioavailability, increased exposure, and decreased clearance. In some cases, the conjugate has increased stability and/or serum half-life relative to ADI alone and/or TNF superfamily ligand alone. In some instances, the stability and/or serum half-life of the conjugate is increased by about or at least about 10%, 20%, 30%, 40%, relative to the ADI alone and/or the TNF superfamily ligand alone. 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% or more.

在一些情况下，相对于单独的ADI和/或单独的TNF超家族配体，缀合物具有增加的生物活性。在一些情况下，相对于单独的ADI和/或单独的TNF超家族配体，缀合物的生物活性增加了约或至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、200％、300％、400％、500％、600％、700％、800％、900％或1000％或更多。在一些情况下，相对于单独的ADI和/或单独的TNF超家族配体，生物活性的增加是协同增加。在一些情况下，相对于单独的ADI和/或单独的TNF超家族配体，生物活性的增加是加和增加。在特定实施例中，生物活性是诱导癌细胞中的细胞死亡或凋亡和/或上调TNF超家族受体表达(例如，死亡受体5(DR5))。在特定情况下，相对于单独的TNF超家族配体，缀合物的ADI组分使TNF超家族配体诱导癌细胞中细胞死亡或凋亡的能力增加了约至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、200％、300％、400％、500％、600％、700％、800％、900％或1000％(例如，通过上调癌细胞上DR5的表达)。In some cases, the conjugate has increased biological activity relative to ADI alone and/or a TNF superfamily ligand alone. In some instances, the biological activity of the conjugate is increased by about or at least about 10%, 20%, 30%, 40%, 50%, 60% relative to ADI alone and/or TNF superfamily ligand alone , 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% or more. In some instances, the increase in biological activity is a synergistic increase relative to the ADI alone and/or the TNF superfamily ligand alone. In some cases, the increase in biological activity is an additive increase relative to ADI alone and/or a TNF superfamily ligand alone. In particular embodiments, the biological activity is induction of cell death or apoptosis in cancer cells and/or upregulation of TNF superfamily receptor expression (eg, death receptor 5 (DR5)). In certain instances, the ADI component of the conjugate increases the ability of the TNF superfamily ligand to induce cell death or apoptosis in cancer cells by about at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% (eg, by upregulating the expression of DR5 on cancer cells).

在特定情况下，相对于单独的ADI和/或单独的TNF超家族配体，缀合物增加了(例如，增加了约至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、200％、300％、400％、500％、600％、700％、800％、900％或1000％或更多)或协同增加了肿瘤细胞杀伤活性和/或凋亡诱导活性。在一些情况下，癌细胞是ADI敏感的癌细胞，或表达低水平或检测不到水平的精氨酸琥珀酸合成酶-1(ASS1)的癌细胞。在特定情况下，癌细胞(例如，ADI敏感癌细胞)选自乳腺癌细胞、肝细胞癌细胞、伯基特淋巴瘤细胞、结肠癌细胞、胶质母细胞瘤癌细胞、白血病细胞、黑色素瘤癌细胞、非小细胞肺癌(NSCLC)细胞、卵巢癌细胞、胰腺癌细胞、前列腺癌细胞和肾癌细胞中的一种或多种。在一些情况下，癌细胞是ADI不敏感或抗ADI癌细胞，或表达相对高水平ASS1的癌细胞。在特定情况下，癌细胞(例如，ADI不敏感的癌细胞)选自乳腺癌细胞、结肠癌细胞和NSCLC细胞中的一种或多种。In certain instances, the conjugate is increased (eg, increased by about at least about 10%, 20%, 30%, 40%, 50%, 60%) relative to ADI alone and/or TNF superfamily ligand alone %, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% or more) or synergistically increased tumor cells Killing activity and/or apoptosis-inducing activity. In some instances, the cancer cells are ADI-sensitive cancer cells, or cancer cells that express low or undetectable levels of argininosuccinate synthase-1 (ASS1). In certain instances, the cancer cells (eg, ADI-sensitive cancer cells) are selected from breast cancer cells, hepatocellular carcinoma cells, Burkitt lymphoma cells, colon cancer cells, glioblastoma cancer cells, leukemia cells, melanoma cells One or more of cancer cells, non-small cell lung cancer (NSCLC) cells, ovarian cancer cells, pancreatic cancer cells, prostate cancer cells, and renal cancer cells. In some cases, the cancer cells are ADI-insensitive or ADI-resistant cancer cells, or cancer cells that express relatively high levels of ASS1. In certain instances, the cancer cells (eg, ADI-insensitive cancer cells) are selected from one or more of breast cancer cells, colon cancer cells, and NSCLC cells.

在一些实施例中，缀合物包含共价连接到三聚体(例如，同源三聚体)多肽的六聚体(例如，同源六聚体)多肽，并且在一些情况下，相对于单独的三聚体多肽，与六聚体多肽的缀合改善了三聚体多肽的物理、药代动力学和/或生物学性质，和/或反之亦然。在一些实施例中，缀合物包括共价连接到第二三聚体(例如，同源三聚体)多肽的第一三聚体(例如，同源三聚体)多肽，并且在一些情况下，相对于单独的第二三聚体多肽，与第一三聚体多肽的缀合改善了第二三聚体多肽的物理、药代动力学和/或生物学性质，和/或反之亦然。在一些情况下，相对于单独的一种或两种组分，缀合物具有改善的物理、药代动力学和/或生物学性质。在特定情况下，相对于单独的一种或两种组分，缀合物具有增加的稳定性和/或血清半衰期，例如，其中相对于单独的一种或两种组分，缀合物的稳定性和/或血清半衰期增加了约至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、200％、300％、400％、500％、600％、700％、800％、900％或1000％或更多。在一些情况下，相对于单独的一种或两种组分，缀合物具有增加的生物活性，例如，其中相对于单独的一种或两种组分，缀合物的生物活性增加了约至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、200％、300％、400％、500％、600％、700％、800％、900％或1000％或更多。In some embodiments, the conjugate comprises a hexameric (eg, homohexameric) polypeptide covalently linked to a trimeric (eg, homotrimeric) polypeptide, and in some cases, relative to The trimeric polypeptide alone, conjugation to the hexameric polypeptide improves the physical, pharmacokinetic and/or biological properties of the trimeric polypeptide, and/or vice versa. In some embodiments, the conjugate includes a first trimeric (eg, homotrimeric) polypeptide covalently linked to a second trimeric (eg, homotrimeric) polypeptide, and in some cases Conjugation to the first trimer polypeptide improves the physical, pharmacokinetic and/or biological properties of the second trimer polypeptide relative to the second trimer polypeptide alone, and/or vice versa Of course. In some cases, the conjugate has improved physical, pharmacokinetic and/or biological properties relative to one or both components alone. In certain instances, the conjugate has increased stability and/or serum half-life relative to one or both components alone, eg, wherein the conjugate has increased stability relative to one or both components alone Stability and/or serum half-life is increased by about at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% , 500%, 600%, 700%, 800%, 900% or 1000% or more. In some cases, the conjugate has increased biological activity relative to one or both components alone, eg, wherein the biological activity of the conjugate is increased relative to one or both components alone by about At least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% or more.

下文对缀合物的单独组分进行了更详细的描述。The individual components of the conjugate are described in more detail below.

精氨酸脱亚胺酶(ADI)某些缀合物包括一种或多种精氨酸脱亚胺酶(ADI)，也称为ADI多肽或ADI酶。在一些实施例中，ADI多肽来自人型支原体、精氨酸支原体、关节炎支原体、海豹脑支原体、猫支原体、海豹支原体、鸽支原体、衣阿华支原体、鳄鱼支原体、短吻鳄支原体、奥氏嗜热盐丝菌或牛支原体。在一些实施例中，ADI多肽来自唾液支原体、泡沫支原体、加拿大支原体、耳支原体、猪滑液支原体、泄殖腔支原体、鹅支原体、产碱支原体、口腔支原体、惰性支原体、火鸡支原体、蜂房支原体、穿透支原体、鸡支原体、梨支原体、类人猿支原体、发酵支原体、狮咽支原体、气管支原体、模仿支原体、乳白色支原体、毛氏支原体、象支原体、肺炎支原体、龟支原体、支原体属CAG:877、或支原体属CAG:472。Arginine Deiminase (ADI) Certain conjugates include one or more arginine deiminase (ADI) enzymes, also known as ADI polypeptides or ADI enzymes. In some embodiments, the ADI polypeptide is from Mycoplasma hominis, Mycoplasma arginine, Mycoplasma arthritis, Mycoplasma cerebellum, Mycoplasma felis, Mycoplasma seal, Mycoplasma pigeon, Mycoplasma Iowa, Mycoplasma crocodile, Mycoplasma alligatorii, Mycoplasma aubergine Thermophilic Halophthora or Mycoplasma bovis. In some embodiments, the ADI polypeptide is from Mycoplasma salivarius, Mycoplasma fronds, Mycoplasma canadensis, Mycoplasma auris, Mycoplasma hyos synovium, Mycoplasma cloaca, Mycoplasma geese, Mycoplasma alkaloids, Mycoplasma oralis, Mycoplasma inerts, Mycoplasma turkeyi, Mycoplasma alpi, Hypermycoplasma, Mycoplasma Gallinarum, Mycoplasma pear, Mycoplasma simian, Mycoplasma fermentum, Mycoplasma leopharynx, Mycoplasma trachea, Mycoplasma mimetic, Mycoplasma opalescent, Mycoplasma trichomes, Mycoplasma elegans, Mycoplasma pneumoniae, Mycoplasma chelae, Mycoplasma CAG:877, or Mycoplasma CAG:472.

下表A1中提供了说明性ADI多肽的氨基酸序列。Amino acid sequences of illustrative ADI polypeptides are provided in Table A1 below.

因此，在一些实施例中，缀合物的ADI组分包括选自表A1(SEQ ID NO:1-68)的氨基酸序列(或其活性变体或片段)，由所述氨基酸序列组成或基本上由所述氨基酸序列组成。活性变体和片段的特定实例包括与选自表A1的序列至少80％、95％、90％、95％、96％、97％、98％或99％相同的氨基酸序列，由所述氨基酸序列组成或基本上由所述氨基酸序列组成。本文其它地方描述了多肽“变体”和“片段”的另外实例。Thus, in some embodiments, the ADI component of the conjugate comprises, consists of, or consists essentially of an amino acid sequence (or an active variant or fragment thereof) selected from Table A1 (SEQ ID NOs: 1-68) consists of the amino acid sequence. Specific examples of active variants and fragments include amino acid sequences that are at least 80%, 95%, 90%, 95%, 96%, 97%, 98%, or 99% identical to sequences selected from Table A1, from which consist of or consist essentially of the amino acid sequence. Additional examples of polypeptide "variants" and "fragments" are described elsewhere herein.

在某些实施例中，ADI具有“ADI活性”，或将精氨酸转化或代谢成瓜氨酸和氨的能力。ADI活性可以根据本领域的常规技术来测量。例如，L-瓜氨酸的量可以通过比色终点测定法(参见例如，Knipp和Vasak，《分析生物化学(Analytical Biochem.)》286:257-264,2000)检测，并与已知量的L-瓜氨酸的标准曲线进行比较，以计算ADI的比活性，所述比活性可以表示为IU/mg的蛋白。在一些实施例中，ADI酶活性的一个IU被定义为在测试的pH和温度下每分钟产生1μmol瓜氨酸的酶的量。In certain embodiments, the ADI has "ADI activity," or the ability to convert or metabolize arginine to citrulline and ammonia. ADI activity can be measured according to routine techniques in the art. For example, the amount of L-citrulline can be detected by a colorimetric end-point assay (see, eg, Knipp and Vasak, Analytical Biochem. 286:257-264, 2000) and compared with known amounts of A standard curve of L-citrulline was compared to calculate the specific activity of ADI, which can be expressed as IU/mg of protein. In some embodiments, one IU of ADI enzymatic activity is defined as the amount of enzyme that produces 1 μmol of citrulline per minute at the pH and temperature tested.

在一些实施例中，ADI是六聚体或同源六聚体ADI，例如，ADI能够在其天然状态下和/或在缀合到缀合物的TNF超家族配体组分时形成六聚体或同源六聚体结构。不受任何一种理论的束缚，假设缀合物的ADI组分的六聚体或同源六聚体结构能够稳定缀合物的TNF超家族配体组分，特别是当后者在其天然状态下和/或在缀合到缀合物的ADI组分时形成三聚体或同源三聚体结构时。六聚体或同源六聚体ADI的特定实例包含衍生自鸽支原体、惰性支原体、鸡支原体和火鸡支原体的天然ADI(例如，分别为SEQ ID NO:9、37、38、50)，以及来自表A1的嵌合ADI(例如，SEQ ID NO:57-68)，包含其活性变体和片段。In some embodiments, the ADI is a hexameric or homohexameric ADI, eg, the ADI is capable of forming a hexamer in its native state and/or when conjugated to the TNF superfamily ligand component of the conjugate Monomeric or homohexameric structures. Without being bound by any one theory, it is hypothesized that the hexameric or homohexameric structure of the ADI component of the conjugate can stabilize the TNF superfamily ligand component of the conjugate, especially when the latter is in its native state and/or when a trimer or homotrimeric structure is formed upon conjugation to the ADI component of the conjugate. Specific examples of hexameric or homohexameric ADIs include native ADIs derived from M. pigeonii, M. inert, M. gallinarum, and M. turkeyi (eg, SEQ ID NOs: 9, 37, 38, 50, respectively), and Chimeric ADIs from Table Al (eg, SEQ ID NOs: 57-68), including active variants and fragments thereof.

本文所述的任何一种或多种ADI多肽可以与本文所述的任何一种或多种TNF超家族配体或三聚体(例如，同源三聚体)多肽结合，以形成缀合物(例如，融合蛋白)。Any one or more ADI polypeptides described herein can be combined with any one or more TNF superfamily ligand or trimeric (eg, homotrimeric) polypeptides described herein to form a conjugate (eg, fusion proteins).

TNF超家族配体某些缀合物包括一种或多种肿瘤坏死因子(TNF)超家族配体，也被称为TNF超家族配体多肽。肿瘤坏死因子受体超家族(TNFRSF)是细胞因子受体的蛋白质超家族，所述蛋白质超家族特征在于通过富含细胞外半胱氨酸的结构域结合肿瘤坏死因子(TNF)的能力。除神经生长因子(NGF)外，所有TNF均与原型TNF-α同源。TNF受体主要涉及细胞凋亡和炎症，但也调节其它信号转导途径，如细胞增殖、存活和分化。术语死亡受体是指含有死亡结构域的TNF受体超家族成员，其实例包含TNFR1、Fas受体、死亡受体4(DR4)以及死亡受体5(DR5)。TNF Superfamily Ligands Certain conjugates include one or more tumor necrosis factor (TNF) superfamily ligands, also known as TNF superfamily ligand polypeptides. The tumor necrosis factor receptor superfamily (TNFRSF) is a protein superfamily of cytokine receptors characterized by the ability to bind tumor necrosis factor (TNF) through an extracellular cysteine-rich domain. With the exception of nerve growth factor (NGF), all TNFs are homologous to the prototype TNF-alpha. TNF receptors are primarily involved in apoptosis and inflammation, but also regulate other signal transduction pathways such as cell proliferation, survival and differentiation. The term death receptor refers to members of the death domain-containing TNF receptor superfamily, examples of which include TNFRl, Fas receptor, death receptor 4 (DR4), and death receptor 5 (DR5).

下表T1中提供了TNF超家族受体和其相应配体的说明性清单。An illustrative list of TNF superfamily receptors and their corresponding ligands is provided in Table T1 below.

因此，在某些实施例中，缀合物的TNF超家族配体组分选自表T1中的配体多肽。在某些实施例中，TNF超家族配体是选自表T1的人多肽配体。Thus, in certain embodiments, the TNF superfamily ligand component of the conjugate is selected from the ligand polypeptides in Table T1. In certain embodiments, the TNF superfamily ligand is a human polypeptide ligand selected from Table T1.

在一些实施例中，所述TNF超家族配体是三聚体或同源三聚体多肽。如上所述，根据一个非限制性理论，缀合物的ADI组分的六聚体或同源六聚体结构能够稳定缀合物的三聚体或同源三聚体TNF超家族配体组分。在某些实施例中，TNF超家族配体是选自表T1的三聚体或同源三聚体多肽配体。In some embodiments, the TNF superfamily ligand is a trimeric or homotrimeric polypeptide. As noted above, according to one non-limiting theory, the hexameric or homohexameric structure of the ADI component of the conjugate is capable of stabilizing the trimeric or homotrimeric TNF superfamily ligand set of the conjugate point. In certain embodiments, the TNF superfamily ligand is a trimeric or homotrimeric polypeptide ligand selected from Table T1.

在一些实施例中，TNF超家族配体诱导癌细胞中的细胞凋亡(例如，通过结合到TNF超家族受体的死亡结构域或死亡受体)。因此，在一些实施例中，TNF超家族配体(例如，三聚体或同源三聚体配体)结合到至少一种TNF死亡受体或含有至少一个死亡结构域的TNF超家族受体。TNF超家族死亡受体的实例包含TNFR1、Fas受体、DR4和DR5。死亡受体配体的特定实例包含TRAIL、TNF-α和FasL。因此，在某些实施例中，缀合物的TNF超家族配体组分选自TRAIL、TNF-α和FasL，任选地人TRAIL、人TNF-α或人FasL中的一种或多种。In some embodiments, the TNF superfamily ligand induces apoptosis in cancer cells (eg, by binding to the death domain or death receptor of a TNF superfamily receptor). Thus, in some embodiments, a TNF superfamily ligand (eg, a trimeric or homotrimeric ligand) binds to at least one TNF death receptor or a TNF superfamily receptor containing at least one death domain . Examples of TNF superfamily death receptors include TNFRl, Fas receptor, DR4 and DR5. Specific examples of death receptor ligands include TRAIL, TNF-alpha and FasL. Thus, in certain embodiments, the TNF superfamily ligand component of the conjugate is selected from TRAIL, TNF-alpha and FasL, optionally one or more of human TRAIL, human TNF-alpha or human FasL .

下表T2中提供了人TRAIL、人TNF-α和人FasL的氨基酸序列。The amino acid sequences of human TRAIL, human TNF-alpha and human FasL are provided in Table T2 below.

在一些实施例中，缀合物的TNF超家族配体组分包括选自表T2(SEQ ID No:69-72)的氨基酸序列(或其活性变体或片段)，由所述氨基酸序列组成或基本上由所述氨基酸序列组成。变体和片段的特定实例包括与选自表T2的序列至少80％、95％、90％、95％、96％、97％、98％或99％相同的氨基酸序列，由所述氨基酸序列组成或基本上由所述氨基酸序列组成。本文其它地方描述了活性多肽“变体”和“片段”的其它实例。In some embodiments, the TNF superfamily ligand component of the conjugate comprises, consists of, an amino acid sequence (or an active variant or fragment thereof) selected from Table T2 (SEQ ID Nos: 69-72) or consist essentially of said amino acid sequence. Specific examples of variants and fragments include amino acid sequences that are at least 80%, 95%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, and consist of, a sequence selected from Table T2 or consist essentially of said amino acid sequence. Other examples of active polypeptide "variants" and "fragments" are described elsewhere herein.

在具体实施例中，缀合物的TNF超家族配体组分是人TNF相关的凋亡诱导配体(TRAIL)多肽，或其变体或片段。TRAIL是由大多数正常组织细胞产生和分泌的细胞因子。它在肿瘤细胞中引起细胞凋亡(例如，通过结合到某些死亡受体)。预测的281氨基酸的TRAIL蛋白具有II型膜蛋白的特征结构，具有单个内部疏水结构域且没有信号序列。TRAIL的细胞外C端结构域与其它TNF家族成员的C端结构域共享22％到28％的同一性。TRAIL与其信号传导受体DR4和DR5之间复合物的形成通过诱导细胞内死亡结构域的寡聚化来触发细胞凋亡。In specific embodiments, the TNF superfamily ligand component of the conjugate is a human TNF-related apoptosis-inducing ligand (TRAIL) polypeptide, or a variant or fragment thereof. TRAIL is a cytokine produced and secreted by most normal tissue cells. It causes apoptosis in tumor cells (eg, by binding to certain death receptors). The predicted 281 amino acid TRAIL protein has the characteristic structure of a type II membrane protein, with a single internal hydrophobic domain and no signal sequence. The extracellular C-terminal domain of TRAIL shares 22% to 28% identity with the C-terminal domains of other TNF family members. Formation of a complex between TRAIL and its signaling receptors DR4 and DR5 triggers apoptosis by inducing oligomerization of the intracellular death domain.

在某些实施例中，缀合物的TRAIL组分包括来自表T2(SEQ ID NO:69和70)的TRAIL序列(或其变体或片段)，由所述氨基酸序列组成或基本上由所述氨基酸序列组成。TRAIL变体的具体实例包含具有以下取代中的任何一个或多个的变体：S96C、S101C、S111C、R170C和K179C。在一些实施例中，TRAIL变体在残基位置具有一组氨基酸取代，其选自Y189Q、R191K、Q193R；H264R、I266L、D267Q；Y189Q、R191K、Q193R；以及Y189Q、R191K、Q193R、I266L中的一个或多个(参见美国申请第2013/0165383号；和第2012/0165267号，其通过引用并入)。TRAIL片段的特定实例包含残基114-281(细胞外结构域)、残基95-281、残基92-281、残基91-281、残基41-281、残基39-281、残基15-281、残基119-281以及全长序列(SEQ ID NO:69)的残基1-281。本文其它地方描述了多肽“变体”和“片段”的另外实例。In certain embodiments, the TRAIL component of the conjugate comprises the TRAIL sequence (or a variant or fragment thereof) from Table T2 (SEQ ID NOs: 69 and 70), consists of or consists essentially of the amino acid sequence composition of the amino acid sequence. Specific examples of TRAIL variants include variants with any one or more of the following substitutions: S96C, S101C, S111C, R170C, and K179C. In some embodiments, the TRAIL variant has a set of amino acid substitutions at residue positions selected from Y189Q, R191K, Q193R; H264R, I266L, D267Q; Y189Q, R191K, Q193R; and Y189Q, R191K, Q193R, I266L one or more (see US Application Nos. 2013/0165383; and 2012/0165267, which are incorporated by reference). Specific examples of TRAIL fragments include residues 114-281 (extracellular domain), residues 95-281, residues 92-281, residues 91-281, residues 41-281, residues 39-281, residues 15-281, residues 119-281, and residues 1-281 of the full-length sequence (SEQ ID NO:69). Additional examples of polypeptide "variants" and "fragments" are described elsewhere herein.

本文所述的任何一种或多种TNF超家族配体可与本文所述的任何一种或多种ADI多肽或六聚体(例如，同源六聚体)多肽组合，以形成缀合物(例如，融合蛋白)。Any one or more of the TNF superfamily ligands described herein can be combined with any one or more of the ADI polypeptides or hexameric (eg, homohexameric) polypeptides described herein to form a conjugate (eg, fusion proteins).

连接子某些缀合物包括一个或多个连接子基团。术语“连接”，“连接子”，“连接子部分”或“L”在本文中用于指可用于将缀合物的一个多肽组分与另一多肽组分分离的连接子，例如，来自TNF超家族配体的ADI多肽，或来自三聚体多肽的六聚体多肽。连接子可以是生理稳定的，或者可以包含可释放的连接子，如不稳定的连接子或可酶促降解的连接子(例如，蛋白水解可切割的连接子)。在某些方面，连接子是肽连接子。在一些方面，连接子非肽连接子或非蛋白质连接子。Linkers Certain conjugates include one or more linker groups. The terms "linker", "linker", "linker moiety" or "L" are used herein to refer to a linker that can be used to separate one polypeptide component of a conjugate from another polypeptide component, eg, ADI polypeptides from TNF superfamily ligands, or hexameric polypeptides from trimeric polypeptides. The linker may be physiologically stable, or may comprise a releasable linker, such as a labile linker or an enzymatically degradable linker (eg, a proteolytically cleavable linker). In certain aspects, the linker is a peptide linker. In some aspects, the linker is not a peptide linker or a non-protein linker.

某些实施例包括一种或多种肽连接子。可以使用本领域的标准技术将此类肽连接子序列掺入缀合物(例如，融合多肽)中。Certain embodiments include one or more peptide linkers. Such peptide linker sequences can be incorporated into conjugates (eg, fusion polypeptides) using standard techniques in the art.

可以基于以下示例性因素选择某些肽连接子序列：(1)其能够采用灵活的扩展构象；(2)其不能采用可以与第一多肽和第二多肽上的功能性表位相互作用的次级结构；(3)其生理稳定性；和(4)缺乏可能与多肽功能性表位或其它特征反应的疏水残基或带电残基。参见例如，George和Heringa,J，《蛋白质工程(Protein Eng.)》15:871-879,2002。在一些实施例中，肽连接子是刚性连接子。在一些实施例中，肽连接子是柔性连接子。在特定实施例中，可以使用能够对肽本身进行建模的计算机程序(Desjarlais和Berg，《美国科学院院报(PNAS.)》90:2256-2260,1993；以及《美国科学院院报》91:11099-11103,1994)或通过噬菌体展示方法合理地设计柔性连接子。Certain peptide linker sequences can be selected based on the following exemplary factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt functional epitopes that can interact with the first and second polypeptides (3) its physiological stability; and (4) lack of hydrophobic or charged residues that may react with functional epitopes or other features of the polypeptide. See, eg, George and Heringa, J, Protein Eng. 15:871-879, 2002. In some embodiments, the peptide linker is a rigid linker. In some embodiments, the peptide linker is a flexible linker. In certain embodiments, computer programs capable of modeling the peptides themselves may be used (Desjarlais and Berg, PNAS. 90:2256-2260, 1993; and PNAS 91: 11099-11103, 1994) or rationally design flexible linkers by phage display methods.

在一些实施例中，肽连接子序列的长度为1到约200个氨基酸。示例性连接子可具有约1-200个氨基酸、1-150个氨基酸、1-100个氨基酸、1-90个氨基酸、1-80个氨基酸、1-70个氨基酸、1-60个氨基酸、1-50个氨基酸、1-40个氨基酸、1-30个氨基酸、1-20个氨基酸、1-10个氨基酸、1-5个氨基酸、1-4个氨基酸、1-3个氨基酸或约1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190或200个或者更多氨基酸的总氨基酸长度。In some embodiments, the peptide linker sequence is 1 to about 200 amino acids in length. Exemplary linkers can have about 1-200 amino acids, 1-150 amino acids, 1-100 amino acids, 1-90 amino acids, 1-80 amino acids, 1-70 amino acids, 1-60 amino acids, 1 -50 amino acids, 1-40 amino acids, 1-30 amino acids, 1-20 amino acids, 1-10 amino acids, 1-5 amino acids, 1-4 amino acids, 1-3 amino acids or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, Total amino acid length of 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 or more amino acids.

肽连接子可以使用如本文别处描述的和本领域已知的任何一种或多种天然存在的氨基酸、一种或多种非天然存在的氨基酸、氨基酸类似物和/或氨基酸模拟物。可以有用地用作连接子的某些氨基酸序列包含在Maratea等人，《基因(Gene)》40:39-46,1985；Murphy等人，《美国科学院院报(PNAS USA.)》83:8258-8262,1986；美国专利第4,935,233号和美国专利第4,751,180号中公开的氨基酸序列。特定的肽连接子序列含有Gly残基、Ser残基和/或Asn残基。如果需要，还可以在肽连接子序列中采用其它近中性氨基酸，如Thr和Ala。Peptide linkers can use any one or more naturally occurring amino acids, one or more non-naturally occurring amino acids, amino acid analogs and/or amino acid mimetics as described elsewhere herein and known in the art. Certain amino acid sequences that may be useful as linkers are contained in Maratea et al., Gene 40:39-46, 1985; Murphy et al., PNAS USA. 83:8258 -8262,1986; amino acid sequences disclosed in US Patent No. 4,935,233 and US Patent No. 4,751,180. Particular peptide linker sequences contain Gly residues, Ser residues and/or Asn residues. Other near-neutral amino acids, such as Thr and Ala, can also be employed in the peptide linker sequence if desired.

下表L1中提供了某些示例性肽连接子。Certain exemplary peptide linkers are provided in Table L1 below.

因此，在某些实施例中，缀合物(例如，融合多肽)包括一种或多种选自表P1的肽连接子。Thus, in certain embodiments, the conjugate (eg, fusion polypeptide) includes one or more peptide linkers selected from Table P1.

在一些实施例中，例如，在非融合或化学连接的缀合物中，连接子为非肽连接子。例如，在一些实施例中，连接子是构建成含有烷基或芳基主链、且含有酰胺、醚、酯、腙、二硫键或其任何组合的有机部分。含有氨基酸、醚和酰胺结合组分的键在生理pH条件下(血清中通常为7.4)是稳定的。还包括含有酯或腙并在血清pH下稳定的键。In some embodiments, eg, in non-fusion or chemically linked conjugates, the linker is a non-peptide linker. For example, in some embodiments, the linker is an organic moiety constructed to contain an alkyl or aryl backbone, and to contain amides, ethers, esters, hydrazones, disulfide bonds, or any combination thereof. Bonds containing amino acid, ether and amide binding components are stable under physiological pH conditions (typically 7.4 in serum). Also included are linkages that contain esters or hydrazones and are stable at serum pH.

在一些情况下，连接子包含增加两个连接原子之间距离的间隔子。间隔子可以进一步增加连接子的柔性和/或长度。间隔子可以包含但不限于烷基、烯基、炔基、芳基、芳烷基、芳烯基、芳炔基；其中每一个可以含有一个或多个杂原子、杂环、氨基酸、核苷酸和糖。In some cases, the linker comprises a spacer that increases the distance between the two linking atoms. Spacers can further increase the flexibility and/or length of the linker. Spacers may include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, aralkyl, aralkenyl, aralkynyl; each of which may contain one or more heteroatoms, heterocycles, amino acids, nucleosides acid and sugar.

在一些实施例中，连接子的长度为约1到约30个原子，或长度为约1、2、3、4、5、6、7、8、9、0、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个原子，包含这其间所有的范围。在某些实施例中，连接子长度为约1到约30个原子，碳链原子可以被独立地选自由O、N或S组成的基团的杂原子取代。在一些实施例中，1到4或5到15个C原子被独立选自O、N、S的杂原子取代。In some embodiments, the linker is about 1 to about 30 atoms in length, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 0, 11, 12, 13, 14 in length , 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 atoms, including all ranges in between. In certain embodiments, the linker is about 1 to about 30 atoms in length, and the carbon chain atoms may be substituted with heteroatoms independently selected from groups consisting of O, N, or S. In some embodiments, 1 to 4 or 5 to 15 C atoms are substituted with heteroatoms independently selected from O, N, S.

在某些实施例中，连接子包括选自以下的结构或由选自以下的结构组成：—O—、—NH—、—S—、—C(O)—、C(O)—NH、NH—C(O)—NH、O—C(O)—NH、—C(S)—、—CH₂—、—CH₂—CH₂—、—CH₂—CH₂—CH₂—、—CH₂—CH₂—CH₂—CH₂—、—O—CH₂—、—CH₂—O—、—O—CH₂—CH₂—、—CH₂—O—CH₂—、—CH₂—CH₂—O—、—O—CH₂—CH₂—CH₂—、—CH₂—O—CH₂—CH₂—、—CH₂—CH₂—O—CH₂—、—CH₂—CH₂—CH₂—O—、—O—CH₂—CH₂—CH₂—CH₂—、—CH₂—O—CH₂—CH₂—CH₂—、—CH₂—CH₂—O—CH₂—CH₂—、—CH₂—CH₂—CH₂—O—CH₂—、—CH₂—CH₂—CH₂—CH₂—O—、—C(O)—NH—CH₂—、—C(O)—NH—CH₂—CH₂—、—CH₂—C(O)—NH—CH₂—、—CH₂—CH₂—C(O)—NH—、—C(O)—NH—CH₂—CH₂—CH₂—、—CH₂—C(O)—NH—CH₂—CH₂—、—CH₂—CH₂—C(O)—NH—CH₂—、—CH₂—CH₂—CH₂—C(O)—NH—、—C(O)—NH—CH₂—CH₂—CH₂—CH₂—、—CH₂—C(O)—NH—CH₂—CH₂—CH₂—、—CH₂—CH₂—C(O)—NH—CH₂—CH₂—、—CH₂—CH₂—CH₂—C(O)—NH—CH₂—、—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—、—CH₂—CH₂—CH₂—CH₂—C(O)—NH—、—NH—C(O)—CH₂—、—CH₂—NH—C(O)—CH₂—、—CH₂—CH₂—NH—C(O)—CH₂—、—NH—C(O)—CH₂—CH₂—、—CH₂—NH—C(O)—CH₂—CH₂、—CH₂—CH₂—NH—C(O)—CH₂—CH₂、—C(O)—NH—CH₂—、—C(O)—NH—CH₂—CH₂—、—O—C(O)—NH—CH₂—、—O—C(O)—NH—CH₂—CH₂—、—NH—CH₂—、—NH—CH₂—CH₂—、—CH₂—NH—CH₂—、—CH₂—CH₂—NH—CH₂—、—C(O)—CH₂—、—C(O)—CH₂—CH₂—、—CH₂—C(O)—CH₂—、—CH₂—CH₂—C(O)—CH₂—、—CH₂—CH₂—C(O)—CH₂—CH₂—、—CH₂—CH₂—C(O)—、—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—、—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—、—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—、二价环烷基、—N(R⁶)—，R⁶是H或选自由烷基、取代的烷基、烯基、取代的烯基、炔基、取代的炔基、芳基和取代的芳基组成的基团的有机基。In certain embodiments, the linker comprises or consists of a structure selected from the group consisting of: -O-, -NH-, -S-, -C(O)-, C(O)-NH, NH—C(O)—NH, O—C(O)—NH,—C(S)—,—CH₂ —, —CH₂ —CH₂ —, —CH₂ —CH₂ —CH₂ —,— CH₂ —CH₂ —CH₂ —CH₂ —, —O—CH₂ —, —CH₂ —O—, —O—CH₂ —CH₂ —, —CH₂ —O—CH₂ —, —CH_2—CH2 —O—, —O—_{CH2—CH2—CH2} —,_—CH2 —O—_CH2—CH2 —,_—CH2—CH2 —O—_CH2 —,_—CH2 — CH₂ —CH₂ —O—, —O—CH₂ —CH₂ —CH₂ —CH₂ —, —CH₂ —O—CH₂ —CH₂ —CH₂ —, —CH₂ —CH₂ —O— CH₂ —CH₂ —, —CH₂ —CH₂ —CH₂ —O—CH₂ —, —CH₂ —CH₂ —CH₂ —CH₂ —O—, —C(O)—NH—CH₂ — ,—C(O)—NH—CH₂ —CH₂ —,—CH₂ —C(O)—NH—CH₂ —,—CH₂ —CH₂ —C(O)—NH—,—C(O )—NH—CH2—_CH2 —_CH2 —,—_CH2 —C(O)—NH—_CH2—CH2 —,—_CH2 —_CH2— C(O)—NH—CH2—, —CH₂ —CH₂ —CH₂ —C(O)—NH—, —C(O)—NH—CH₂ —CH₂ —CH₂ —CH₂ —, —CH₂ —C(O)—NH— CH₂ —CH₂ —CH₂ —, —CH₂ —CH₂ —C(O)—NH—CH₂ —CH₂ —, —CH₂ —CH₂ —CH₂ —C(O)—NH—CH₂ —, —CH₂ —CH₂ —CH₂ —C(O)—NH—CH₂ —CH₂ —, —CH₂ —CH₂ —CH₂ —CH₂ —C(O)—NH—, —NH— C(O)—CH₂ —, —CH₂ —NH—C(O)—CH₂ —, —CH₂ —CH₂ —NH—C(O)—CH₂ —, —NH—C(O)— CH₂ —CH₂ —, —CH₂ —NH—C(O)—CH₂ —CH₂ , —CH₂ —CH₂ —NH—C(O)—CH₂ —CH₂ 、—C(O)—NH—CH₂ —,—C(O)—NH—CH₂ —CH₂ —,—O—C(O)—NH—CH₂ —,—O—C(O) —NH—CH₂ —CH₂ —, —NH—CH₂ —, —NH—CH₂ —CH₂ —, —CH₂ —NH—CH₂ —, —CH 2 —CH 2 —NH—CH 2 —, —CH₂ —CH₂ —NH—CH₂ —, —C(O)—CH₂ —, —C(O)—CH₂ —CH₂ —, —CH₂ —C(O)—CH₂ —, —CH₂ —CH₂ —C(O)—CH₂ —, —CH₂ —CH₂ —C(O)—CH₂ —CH₂ —, —CH₂ —CH₂ —C(O)—, —CH₂ —CH₂ —CH₂ —C(O)—NH —CH₂ —CH₂ —NH—, —CH₂ —CH₂ —CH₂ —C(O)—NH—CH₂ —CH₂ —NH—C(O)—, —CH₂ —CH₂ —CH₂ —C(O)—NH—CH₂ —CH₂ —NH—C(O)—CH₂ —, divalent cycloalkyl, —N(R⁶ )—, R⁶ is H or selected from alkyl, substituted The organic group of the group consisting of alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl and substituted aryl.

在一些实施例中，连接子是稳定连接子。在一些实施例中，稳定连接子选自由以下组成的基团：琥珀酰亚胺、丙酸、羧甲基连接、醚、氨基甲酸酯、酰胺、胺、脲、酰亚胺、脂族C-C键和硫醚。在一些实施例中，连接子基团是亲水性的，例如，以增强缀合物在体液中的溶解度。In some embodiments, the linker is a stable linker. In some embodiments, the stabilizing linker is selected from the group consisting of: succinimide, propionic acid, carboxymethyl linkage, ether, carbamate, amide, amine, urea, imide, aliphatic C-C bond and thioether. In some embodiments, the linker group is hydrophilic, eg, to enhance the solubility of the conjugate in body fluids.

在一些实施例中，连接子包括诸如聚乙二醇或聚丙二醇等聚合物，或者由所述聚合物组成。本文所用的术语“PEG”、“聚乙二醇”和“聚(乙二醇)”是可互换的，并且意指包括任何水溶性聚(环氧乙烷)衍生物。PEG是众所周知的聚合物，在许多水性和有机溶剂中具有良好的溶解性，其表现出低毒性，缺乏免疫原性，并且其透明、无色、无味且稳定。如本文所述，可以用其它水溶性聚合物获得类似产物，包含但不限于：聚乙烯醇、其它聚(烯化氧)(如聚(丙二醇)等)、聚(氧乙基化多元醇)(如聚(氧乙基化甘油)等)、羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、聚-1,3-二氧戊环、聚-l,3,6-三恶烷、乙烯/马来酸酐以及聚氨基酸。本领域技术人员将能够基于所需的剂量、循环时间、抗蛋白水解性和其它考虑因素来选择所需的聚合物。In some embodiments, the linker includes or consists of a polymer such as polyethylene glycol or polypropylene glycol. As used herein, the terms "PEG", "polyethylene glycol" and "poly(ethylene glycol)" are interchangeable and are meant to include any water-soluble poly(ethylene oxide) derivative. PEG is a well-known polymer with good solubility in many aqueous and organic solvents, it exhibits low toxicity, lacks immunogenicity, and it is transparent, colorless, odorless, and stable. Similar products can be obtained with other water-soluble polymers, as described herein, including, but not limited to: polyvinyl alcohol, other poly(alkylene oxides) (eg, poly(propylene glycol), etc.), poly(oxyethylated polyols) (such as poly(oxyethylated glycerol), etc.), carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6- Trioxane, ethylene/maleic anhydride and polyamino acids. Those skilled in the art will be able to select the desired polymer based on the desired dosage, cycle time, resistance to proteolysis, and other considerations.

通常，根据本文所述的缀合物使用的PEG包括以下结构：“-(OCH2CH2)n-”，其中(n)为约1到4000、约20到1400、或约20-800。在特定实施例中，PEG还包含“-O-(CH2CH2O)n-CH2CH2-”和“-(OCH2CH2)n-O-”，这取决于末端氧是否已被置换。术语“PEG”包含具有各种末端或“封端”基团的结构。术语“PEG”还包括含有大部分(即大于50％)-OCH2CH2-重复亚单元的聚合物。关于具体形式，PEG可以采用任何数量的各种分子量，以及结构或几何形状，例如“分支的”、“线性的”、“分叉的”、“多功能的”PEG分子。Typically, PEGs used in accordance with the conjugates described herein include the structure: "-(OCH2CH2)n-", wherein (n) is about 1 to 4000, about 20 to 1400, or about 20-800. In certain embodiments, the PEG further comprises "-O-(CH2CH2O)n-CH2CH2-" and "-(OCH2CH2)n-O-", depending on whether the terminal oxygen has been replaced. The term "PEG" encompasses structures having various terminal or "capped" groups. The term "PEG" also includes polymers containing a majority (ie, greater than 50%) of -OCH2CH2- repeating subunits. Regarding specific forms, PEG can take any number of various molecular weights, as well as structures or geometries, such as "branched", "linear", "forked", "multifunctional" PEG molecules.

在以下文献中描述了代表性的聚合试剂和用于将这种聚合物缀合到活性部分的方法：Harris,J.M.和Zalipsky,S.编辑，《聚(乙二醇)化学和生物应用(Poly(ethyleneglycol),Chemistry and Biological Applications)》，ACS，华盛顿，1997；Veronese,F.和J.M.Harris编辑，《肽和蛋白质聚乙二醇化(Peptide and Protein PEGylation)》，《先进药物输送评论(Advanced Drug Delivery Reviews)》，54(4)；453-609(2002)；Zalipsky,S.等人，《聚乙二醇化学：生物技术和生物医学应用(Polyethylene Glycol Chemistry:Biotechnical and Biomedical Applications)》中的“功能化聚(乙二醇)在多肽修饰中的应用”，J.M.Harris编辑，Plenus出版社，纽约(1992)；Zalipsky(1995)，《先进药物评论(Advanced Drug Reviews)》16:157-182；以及Roberts等人，《先进药物输送评论》54,459-476(2002)。Representative polymerization reagents and methods for conjugating such polymers to reactive moieties are described in: Harris, J.M. and Zalipsky, S. eds., "Poly(ethylene glycol) chemical and biological applications (Poly (ethyleneglycol), Chemistry and Biological Applications), ACS, Washington, 1997; Veronese, F. and J.M. Harris, eds., Peptide and Protein PEGylation, Advanced Drug Delivery Review Delivery Reviews), 54(4); 453-609 (2002); Zalipsky, S. et al., Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications "The use of functionalized poly(ethylene glycol) in polypeptide modification," edited by J.M. Harris, Plenus Press, New York (1992); Zalipsky (1995), Advanced Drug Reviews 16:157-182 ; and Roberts et al., Advanced Drug Delivery Review 54, 459-476 (2002).

多种PEG衍生物都是可商购的并且适用于制备本公开的PEG-缀合物。例如，NOF公司的系列提供了许多PEG衍生物，包含甲氧基聚乙二醇和活化的PEG衍生物(如琥珀酰亚胺酯、甲氧基-PEG胺、马来酰亚胺和羧酸)，用于通过各种方法偶联到多肽和多核苷酸，并且内克塔治疗(Nektar Therapeutics)的高级聚乙二醇化还提供多种PEG偶联技术，以提高治疗的安全性和疗效。用于形成缀合物的另外的PEG包含可从Polypure(挪威)、QuantaBioDesign LTD(俄亥俄州)、JenKem Technology、Nanocs Corporation和Sunbio，Inc(南韩)获得的PEG。在例如Pasut等人，《治疗术专利专家评论(ExpertOpin.Ther.Patents.)》14(6)859-893,2004中描述了适用于形成缀合物的另外的PEG试剂和缀合方法。A variety of PEG derivatives are commercially available and suitable for use in preparing the PEG-conjugates of the present disclosure. For example, NOF's The series offers a number of PEG derivatives, including methoxy polyethylene glycols and activated PEG derivatives (such as succinimidyl esters, methoxy-PEG amines, maleimides, and carboxylic acids), for use through various A variety of methods are available for coupling to polypeptides and polynucleotides, and Nektar Therapeutics' advanced PEGylation also offers a variety of PEG conjugation techniques to improve the safety and efficacy of the treatment. Additional PEGs used to form the conjugates include PEGs available from Polypure (Norway), QuantaBioDesign LTD (Ohio), JenKem Technology, Nanocs Corporation and Sunbio, Inc (South Korea). Additional PEG reagents and conjugation methods suitable for forming conjugates are described, for example, in Pasut et al., Expert Opin. Ther. Patents. 14(6) 859-893, 2004.

在以下专利中描述了线性或分支的PEG聚合物和其衍生物或缀合物的制备：例如，美国专利第4,904,584号；第5,428,128号；第5,621,039号；第5,622,986号；第5,643,575号；第5,728,560号；第5,730,990号；第5,738,846号；第5,811,076号；第5,824,701号；第5,840,900号；第5,880,131号；第5,900,402号；第5,902,588号；第5,919,455号；第5,951,974号；第5,965,119号；第5,965,566号；第5,969,040号；第5,981,709号；第6,011,042号；第6,042,822号；第6,113,906号；第6,127,355号；第6,132,713号；第6,177,087号；第6,180,095号；第6,448,369号；第6,495,659号；第6,602,498号；第6,858,736号；第6,828,401号；第7,026,440号；第7,608,678号；第7,655,747号；第7,786,221号；第7,872,072号；以及第7,910,661号，这些专利中的每一个通过引用以其全文并入本文。The preparation of linear or branched PEG polymers and derivatives or conjugates thereof is described in, for example, US Pat. Nos. 4,904,584; 5,428,128; 5,621,039; 5,622,986; 5,643,575; 5,728,560 No. 5,730,990; No. 5,738,846; No. 5,811,076; No. 5,824,701; No. 5,840,900; No. 5,880,131;第5,969,040号；第5,981,709号；第6,011,042号；第6,042,822号；第6,113,906号；第6,127,355号；第6,132,713号；第6,177,087号；第6,180,095号；第6,448,369号；第6,495,659号；第6,602,498号；第6,858,736 6,828,401; 7,026,440; 7,608,678; 7,655,747; 7,786,221; 7,872,072; and 7,910,661, each of which is hereby incorporated by reference in its entirety.

在某些实施例中，前述连接子可任选。In certain embodiments, the aforementioned linkers are optional.

多肽变体某些实施例包含本文所述参考序列的“变体”和“片段”，无论是通过名称还是通过参考表或序列标识符描述。实例包含本文所述的任何ADI多肽、TNF超家族配体多肽和融合多肽。“变体”序列是指在一个或多个取代、缺失(例如，截短)、添加和/或插入上与参考序列不同的多肽或多核苷酸序列。变体多肽具有生物学活性，即，其继续具有参考多肽的酶促或结合活性。此类变体可以由例如遗传多态性和/或人为操纵产生。Polypeptide Variants Certain embodiments include "variants" and "fragments" of the reference sequences described herein, whether described by name or by reference to a table or sequence identifier. Examples include any of the ADI polypeptides, TNF superfamily ligand polypeptides, and fusion polypeptides described herein. A "variant" sequence refers to a polypeptide or polynucleotide sequence that differs from a reference sequence in one or more substitutions, deletions (eg, truncations), additions, and/or insertions. A variant polypeptide has biological activity, ie, it continues to have the enzymatic or binding activity of the reference polypeptide. Such variants can result from, for example, genetic polymorphism and/or human manipulation.

在许多情况下，生物活性变体将含有一个或多个保守取代。“保守取代”是氨基酸取代具有相似性质的另一种氨基酸的取代，使得肽化学领域的技术人员预期多肽的二级结构和亲水性质基本上不变。如上所述，可以对本公开的多核苷酸和多肽的结构进行修饰，并且仍然获得编码具有期望特征的变体或衍生多肽的功能分子。当期望改变多肽的氨基酸序列以产生本文所述的多肽的等同物或甚至改进的变体或部分时，本领域技术人员通常会改变编码DNA序列的一个或多个密码子。In many cases, biologically active variants will contain one or more conservative substitutions. A "conservative substitution" is a substitution of an amino acid for another amino acid of similar properties such that the secondary structure and hydrophilic properties of the polypeptide are expected to be substantially unchanged by those skilled in the art of peptide chemistry. As noted above, the structures of the polynucleotides and polypeptides of the present disclosure can be modified and still obtain functional molecules that encode variant or derived polypeptides having desired characteristics. When it is desired to alter the amino acid sequence of a polypeptide to produce equivalents or even improved variants or portions of the polypeptides described herein, one of skill in the art will typically alter one or more codons encoding the DNA sequence.

例如，某些氨基酸可以取代蛋白质结构中的其它氨基酸，而不会明显丧失相互作用的结合能力。由于蛋白质的相互作用能力和性质决定了蛋白质的生物功能活性，因此可以在蛋白质序列中进行某些氨基酸序列取代，当然，也可以在其基础DNA编码序列中进行，但仍然可以获得具有类似性质的蛋白质。因此预期可以对所公开组合物的肽序列或编码所述肽的相应DNA序列进行各种改变而不会明显丧失其效用。For example, certain amino acids can be substituted for other amino acids in the protein structure without appreciably losing the binding capacity of the interaction. Since the ability and nature of protein interactions determine the biological functional activity of the protein, certain amino acid sequence substitutions can be made in the protein sequence and, of course, in its underlying DNA coding sequence, but it is still possible to obtain a protein with similar properties. protein. It is therefore contemplated that various changes may be made to the peptide sequences of the disclosed compositions or the corresponding DNA sequences encoding the peptides without significant loss of utility.

在进行这种改变时，可以考虑氨基酸的亲水指数。亲水氨基酸指数在赋予蛋白质相互作用生物学功能方面的重要性在本领域中公知(Kyte和Doolittle，1982，通过引用并入本文)。可以理解的是，氨基酸的相对亲水特性有助于所得蛋白质的二级结构，这又定义了蛋白质与其它分子，例如，酶、底物、受体、DNA、抗体、抗原等的相互作用。根据氨基酸的疏水性和电荷特征，每种氨基酸都被指定了亲水指数(Kyte和Doolittle，1982)。这些数值是：异亮氨酸(+4.5)；缬氨酸(+4.2)；亮氨酸(+3.8)；苯丙氨酸(+2.8)；半胱氨酸(+2.5)；蛋氨酸(+1.9)；丙氨酸(+1.8)；甘氨酸(–0.4)；苏氨酸(–0.7)；丝氨酸(–0.8)；色氨酸(–0.9)；酪氨酸(–1.3)；脯氨酸(–1.6)；组氨酸(–3.2)；谷氨酸盐(–3.5)；谷氨酰胺(–3.5)；天冬氨酸盐(–3.5)；天冬酰胺(–3.5)；赖氨酸(–3.9)；以及精氨酸(–4.5)。本领域已知某些氨基酸可以被具有相似亲水指数或分数的其它氨基酸取代，并仍然产生具有相似生物活性的蛋白质，即，仍然获得生物学功能等同的蛋白质。在进行此类改变时，优选亲水指数在±2内的氨基酸取代，特别优选在±1内的氨基酸取代，并且甚至更加特别优选在±0.5内的氨基酸取代。In making such changes, the hydropathic index of the amino acid can be considered. The importance of the hydropathic amino acid index in conferring biological function on protein interactions is well known in the art (Kyte and Doolittle, 1982, incorporated herein by reference). It is understood that the relative hydrophilic properties of amino acids contribute to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules, eg, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: Isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine (+2.5); Methionine (+ 1.9); Alanine (+1.8); Glycine (–0.4); Threonine (–0.7); Serine (–0.8); Tryptophan (–0.9); Tyrosine (–1.3); Proline (–1.6); histidine (–3.2); glutamate (–3.5); glutamine (–3.5); aspartate (–3.5); asparagine (–3.5); lysine acid (–3.9); and arginine (–4.5). It is known in the art that certain amino acids can be substituted with other amino acids having similar hydropathic indices or scores and still yield proteins with similar biological activity, ie, still obtain biologically functionally equivalent proteins. In making such changes, amino acid substitutions with a hydropathic index within ±2 are preferred, amino acid substitutions within ±1 are particularly preferred, and amino acid substitutions within ±0.5 are even more particularly preferred.

在本领域还应理解可以基于亲水性来有效地进行相似氨基酸的取代。美国专利4,554,101(特别通过引用以其全文并入本文)指出，由蛋白质相邻氨基酸的亲水性支配的其最大局部平均亲水性与蛋白质的生物学性质相关。如美国专利4,554,101中所详述的，以下亲水性值已指定为氨基酸残基：精氨酸(+3.0)；赖氨酸(+3.0)；天冬氨酸(+3.0±1)；谷氨酸(+3.0±1)；丝氨酸(+0.3)；天冬酰胺(+0.2)；谷氨酰胺(+0.2)；甘氨酸(0)；苏氨酸(-0.4)；脯氨酸(-0.5±1)；丙氨酸(-0.5)；组氨酸(-0.5)；半胱氨酸(-1.0)；蛋氨酸(-1.3)；缬氨酸(-1.5)；亮氨酸(-1.8)；异亮氨酸(-1.8)；酪氨酸(-2.3)；苯丙氨酸(-2.5)；色氨酸(-3.4)。应当理解的是，氨基酸可以取代具有相似亲水性质的另一种氨基酸，并且仍然获得生物学上等同的，特别是免疫学上等同的蛋白质。在此类改变中，优选亲水性值在±2内的氨基酸取代，特别优选在±1内的氨基酸取代并且甚至更加特别优选在±0.5内的氨基酸取代。It is also understood in the art that similar amino acid substitutions can be made efficiently based on hydrophilicity. US Patent 4,554,101, specifically incorporated herein by reference in its entirety, states that its maximum local average hydrophilicity, governed by the hydrophilicity of adjacent amino acids of a protein, correlates with the biological properties of the protein. As detailed in US Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); Serine (+3.0±1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 ±1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8) ; Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4). It will be appreciated that an amino acid can be substituted for another amino acid having similar hydrophilic properties and still obtain a biologically equivalent, especially immunologically equivalent protein. Among such changes, amino acid substitutions with hydrophilicity values within ±2 are preferred, amino acid substitutions within ±1 are particularly preferred and amino acid substitutions within ±0.5 are even more particularly preferred.

如上所述，氨基酸取代因此通常基于氨基酸侧链取代基的相对相似性，例如，其疏水性、亲水性、电荷、大小等。考虑到各种前述特征的示例性取代是本领域技术人员公知的，并且包含：精氨酸和赖氨酸；谷氨酸盐和天冬氨酸盐；丝氨酸和苏氨酸；谷氨酰胺和天冬酰胺；以及缬氨酸、亮氨酸和异亮氨酸。As noted above, amino acid substitutions are therefore generally based on the relative similarity of amino acid side chain substituents, eg, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into account the various aforementioned features are well known to those skilled in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

可以基于残基的极性、电荷、溶解度、疏水性、亲水性和/或两亲性质的相似性进一步进行氨基酸取代。例如，带负电荷的氨基酸包含天冬氨酸和谷氨酸；带正电荷的氨基酸包含赖氨酸和精氨酸；以及具有相似亲水性数值的不带电极性头部基团的氨基酸包含亮氨酸、异亮氨酸和缬氨酸；甘氨酸和丙氨酸；天冬酰胺和谷氨酰胺；以及丝氨酸、苯丙氨酸和酪氨酸。可以代表保守变化的其它氨基酸基团包含：(1)ala、pro、gly、glu、asp、gln、asn、ser、thr；(2)cys、ser、tyr、thr；(3)val、ile、leu、met、ala、phe；(4)lys、arg、his；以及(5)phe、tyr、trp、his。Further amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic properties of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with similar hydrophilicity values without a polar head group include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; and serine, phenylalanine, and tyrosine. Other amino acid groups that can represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.

变体还可以或可替代地含有非保守变化。在优选的实施例中，变体多肽与天然或参考序列的不同之处在于取代、缺失或添加少于约10、9、8、7、6、5、4、3、2个氨基酸，或甚至1个氨基酸。变体也可以(或可替代地)通过例如对多肽的免疫原性、二级结构、酶活性和/或亲水性质影响最小的氨基酸的缺失或添加进行修饰。Variants may also or alternatively contain non-conservative changes. In preferred embodiments, variant polypeptides differ from the native or reference sequence by substitution, deletion or addition of less than about 10, 9, 8, 7, 6, 5, 4, 3, 2 amino acids, or even 1 amino acid. Variants may also (or alternatively) be modified by, for example, deletion or addition of amino acids that minimally affect the immunogenicity, secondary structure, enzymatic activity and/or hydrophilic properties of the polypeptide.

在某些实施例中，多肽序列长度为约、至少约、或多至约5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、55、60、65、70、75、80、85、90、95、100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、290、300、310、320、330、340、350、360、370、380、390、400、410、420、430、440、450、460、470、480、490、500、510、520、530、540、550、560、570、580、590、600、610、620、630、640、650、660、670、680、690、700、710、720、730、740、750、760、770、780、790、800、800、810、820、830、840、850、860、870、880、890、900、900、910、920、930、940、950、960、970、980、990、1000或更多个连续氨基酸(包含其间的所有整数)，并且可以包括参考序列的全部或一部分(参见例如表或序列表)。In certain embodiments, the polypeptide sequence is about, at least about, or up to about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 or more consecutive amino acids (including all integers therebetween), and may include all or a portion of a reference sequence (see, eg, Table or Sequence Listing).

在一些实施例中，多肽序列由约或不超过约5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、55、60、65、70、75、80、85、90、95、100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、290、300、310、320、330、340、350、360、370、380、390、400、410、420、430、440、450、460、470、480、490、500、510、520、530、540、550、560、570、580、590、600、610、620、630、640、650、660、670、680、690、700、710、720、730、740、750、760、770、780、790、800、800、810、820、830、840、850、860、870、880、890、900、900、910、920、930、940、950、960、970、980、990、1000或更多个连续氨基酸(包含其间的所有整数)组成，并且可以包括参考序列的全部或部分(参见例如表或序列表)。In some embodiments, the polypeptide sequence consists of about or not more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 or more contiguous amino acids (including all integers therebetween) and may include all or part of a reference sequence (see, eg, Table or Sequence Listing).

在某些实施例中，多肽序列约为10-1000、10-900、10-800、10-700、10-600、10-500、10-400、10-300、10-200、10-100、10-50、10-40、10-30、10-20、20-1000、20-900、20-800、20-700、20-600、20-500、20-400、20-300、20-200、20-100、20-50、20-40、20-30、50-1000、50-900、50-800、50-700、50-600、50-500、50-400、50-300、50-200、50-100、100-1000、100-900、100-800、100-700、100-600、100-500、100-400、100-300、100-200、200-1000、200-900、200-800、200-700、200-600、200-500、200-400或200-300个连续的氨基酸(包含其间的所有范围)，并且可以包含参考序列的全部或部分。在某些实施例中，任何参考多肽的C端或N端区域可被截短约1、2、3、4、5、6、7、8、9、10、15、20、25、30、35、40、45、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200、250、300、350、400、450、500、550、600、650、700、750、或800或更多个氨基酸，或约10-50、20-50、50-100、100-150、150-200、200-250、250-300、300-350、350-400、400-450、450-500、500-550、550-600、600-650、650-700、700-750、750-800或更多个氨基酸(包含其间的所有整数和范围(例如，101、102、103、104、105))，只要截短的多肽保留参考多肽的结合特性和/或活性。通常，生物活性片段具有不少于其衍生自的生物活性参考多肽的活性的约1％、约5％、约10％、约25％或约50％。In certain embodiments, the polypeptide sequence is about 10-1000, 10-900, 10-800, 10-700, 10-600, 10-500, 10-400, 10-300, 10-200, 10-100 , 10-50, 10-40, 10-30, 10-20, 20-1000, 20-900, 20-800, 20-700, 20-600, 20-500, 20-400, 20-300, 20 -200, 20-100, 20-50, 20-40, 20-30, 50-1000, 50-900, 50-800, 50-700, 50-600, 50-500, 50-400, 50-300 , 50-200, 50-100, 100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1000, 200 -900, 200-800, 200-700, 200-600, 200-500, 200-400, or 200-300 contiguous amino acids (including all ranges therebetween), and may encompass all or part of a reference sequence. In certain embodiments, the C-terminal or N-terminal region of any reference polypeptide can be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 or more amino acids, or about 10-50, 20-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300- 350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800 or more amino acids (including all integers and ranges therebetween) (eg, 101, 102, 103, 104, 105)), so long as the truncated polypeptide retains the binding properties and/or activity of the reference polypeptide. Typically, a biologically active fragment has no less than about 1%, about 5%, about 10%, about 25%, or about 50% of the activity of the biologically active reference polypeptide from which it is derived.

在某些情况下，变体将显示与参考多肽序列至少约30％、40％、50％、55％、60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的相似性或序列同一性或序列同源性。此外，设想序列与天然或亲本序列的差异在于添加(例如，C端添加、N端添加、两者)、缺失、截短、插入或取代了(例如，保守取代)约1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、55、60、65、70、75、80、85、90、95、或100个氨基酸(包含其间的所有整数和范围)，但保留了亲本或参考多肽序列的特性或活性。In certain instances, a variant will exhibit at least about 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91% identical to the reference polypeptide sequence %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity or sequence identity or sequence homology. Furthermore, it is contemplated that the sequence differs from the native or parental sequence by additions (eg, C-terminal additions, N-terminal additions, both), deletions, truncations, insertions, or substitutions (eg, conservative substitutions) by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids (including all integers and ranges therebetween), but retain the properties or activity of the parental or reference polypeptide sequence.

在一些实施例中，变体多肽与参考序列的差异在于至少一个但少于50、40、30、20、15、10、8、6、5、4、3或2个氨基酸残基。在某些实施例中，变体多肽与参考序列的差异在于至少1％但小于20％、15％、10％或5％的残基。(如果此比较要求比对，则应比对序列以获得最大相似性。删除或插入或错配的“环”序列被视为差异。)In some embodiments, the variant polypeptide differs from the reference sequence by at least one but less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3, or 2 amino acid residues. In certain embodiments, the variant polypeptide differs from the reference sequence by at least 1% but less than 20%, 15%, 10% or 5% of residues. (If this comparison requires alignment, the sequences should be aligned for maximum similarity. Deleted or inserted or mismatched "loop" sequences are considered differences.)

序列之间的序列相似性或序列同一性的计算(这些术语在本文中可互换使用)如下进行。为了测定两个氨基酸序列、或两个核酸序列的百分比同一性，出于最佳比较的目的将序列比对(例如，可以在第一和第二氨基酸或核酸序列中的一个或两个中引入空位以用于最佳比对，并且出于比较目的，非同源序列可以忽略)。在某些实施例中，出于比较目的比对的参考序列长度是参照序列长度的至少30％、优选地至少40％、更优选地至少50％、60％、以及更甚优选地70％、80％、90％、100％。然后比较对应的氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置被与在第二序列中的相应位置相同的氨基酸残基或核苷酸占据时，则这些分子在那个位置是一致的。Calculation of sequence similarity or sequence identity between sequences (these terms are used interchangeably herein) is performed as follows. To determine the percent identity of two amino acid sequences, or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (eg, can be introduced in one or both of the first and second amino acid or nucleic acid sequences Gap for optimal alignment and non-homologous sequences can be ignored for comparison purposes). In certain embodiments, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably 70% of the length of the reference sequence, 80%, 90%, 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.

两个序列之间的百分比同一性是所述序列共享的相同位置的数量的函数，考虑了空位数量以及每个空位的长度，需要引入它们以用于两个序列的最佳比对。The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

序列比较和两个序列之间百分比同一性的测定可以使用数学算法来完成。在一个优选的实施例中，利用Needleman和Wunsch(《分子生物学杂志(J.Mol.Biol.)》48:444-453,1970)算法使用Blossum 62矩阵或PAM250矩阵，空位权重16、14、12、10、8、6、或4以及长度权重1、2、3、4、5、或6来测定两个氨基酸序列之间的百分比同一性，所述算法已并入GCG软件包中的GAP程序中。在又另一优选的实施例中，利用GCG软件包中的GAP程序，使用NWSgapdnCMP矩阵和空位权重40、50、60、70或80以及长度权重1、2、3、4、5或6来测定两个核苷酸序列之间的百分比同一性。一组特别优选的参数(除非另有说明，否则应使用的参数)是Blossum62评分矩阵，空位罚分为12，空位延伸罚分为4，并且移码空位罚分为5。Comparison of sequences and determination of percent identity between two sequences can be accomplished using mathematical algorithms. In a preferred embodiment, the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453, 1970) is used to use the Blossum 62 matrix or the PAM250 matrix, with vacancy weights 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 to determine percent identity between two amino acid sequences, the algorithm has been incorporated into GAP in the GCG software package program. In yet another preferred embodiment, the NWSgapdnCMP matrix and gap weights of 40, 50, 60, 70 or 80 and length weights of 1, 2, 3, 4, 5 or 6 are used to determine the GAP program in the GCG software package The percent identity between two nucleotide sequences. A particularly preferred set of parameters (which should be used unless otherwise stated) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

可以利用已合并在ALIGN程序(版本2.0)中的E.Meyers和W.Miller(《计算机在生物科学中的应用(Cabios)》4:11-17,1989)的算法，使用PAM120权重残基表、空位长度罚分12以及空位罚分4来测定两个氨基酸或核苷酸序列之间的百分比同一性。The algorithm of E. Meyers and W. Miller (Cabios 4:11-17, 1989) has been incorporated into the ALIGN program (version 2.0) using the PAM120 weight residue table , a gap length penalty of 12, and a gap penalty of 4 to determine the percent identity between two amino acid or nucleotide sequences.

本文描述的核酸和蛋白质序列可以用作“查询序列”以对公共数据库进行搜索，从而例如，鉴定其它家族成员或相关序列。可以使用Altschul等人，(1990，《分子生物学杂志(J.Mol.Biol.)》215:403-10)的NBLAST和XBLAST程序(版本2.0)来进行这种搜索。可以用NBLAST程序(得分＝100，字长＝12)进行BLAST核苷酸搜索，以获得与本文描述的核酸分子同源的核苷酸序列。可以用XBLAST程序(得分＝50，字长＝3)进行BLAST蛋白质搜索，以获得与本文描述的蛋白质分子同源的氨基酸序列。为了获得用于比较目的的有空位的比对，可以利用如在Altschul等人，(《核酸研究(Nucleic Acids Res)》25:3389-3402，1997)中所描述的空位BLAST。当利用BLAST和空位BLAST程序时，可以使用相应程序(例如，XBLAST和NBLAST)的默认参数。The nucleic acid and protein sequences described herein can be used as "query sequences" to conduct searches of public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990, J. Mol. Biol. 215:403-10). BLAST nucleotide searches can be performed with the NBLAST program (score=100, wordlength=12) to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program (score=50, wordlength=3) to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul et al., (Nucleic Acids Res 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the corresponding programs (eg, XBLAST and NBLAST) can be used.

在一些实施例中，如上所述，可以使用BLAST比对工具评估多核苷酸和/或多肽。局部比对仅由一对序列区段组成，每个序列区段中的一个序列区段被比较。对Smith-Waterman或Sellers算法的修正将找到所有不能通过扩展或修剪来改善分数的称为高分值区段对(HSP)的片段对。BLAST比对的结果包含统计学测量，以指示仅偶然可以预期BLAST分数的可能性。In some embodiments, polynucleotides and/or polypeptides can be assessed using a BLAST alignment tool, as described above. A local alignment consists of only a pair of sequence segments, one of each sequence segment being compared. A modification of the Smith-Waterman or Sellers algorithm will find all pairs of segments called high scoring segment pairs (HSPs) that cannot be extended or trimmed to improve the score. The results of BLAST alignments contain statistical measures to indicate the likelihood that a BLAST score could be expected by chance only.

根据与每个比对序列相关的空位和取代的数量计算原始分数S，其中较高的相似性分数表示更显著的比对。取代分数由查询表给出(参见PAM，BLOSUM)。A raw score S is calculated based on the number of gaps and substitutions associated with each aligned sequence, where higher similarity scores indicate more significant alignments. Substitution scores are given by look-up tables (see PAM, BLOSUM).

空位分数通常被计算为G(空位开放罚分)和L(空位延伸罚分)之和。对于长度为n的空位，空位成本为G+Ln。空位成本G和L的选择是经验性的，但习惯上选择G(10-15)的高值，例如11，以及L(1-2)的低值，例如，1。The gap score is usually calculated as the sum of G (gap opening penalty) and L (gap extension penalty). For a vacancy of length n, the vacancy cost is G+Ln. The choice of gap costs G and L is empirical, but it is customary to choose a high value of G(10-15), eg, 11, and a low value of L(1-2), eg, 1.

比特分数S'衍生自原始比对分数S，其中已经考虑了所使用的评分系统的统计特性。比特分数相对于评分系统被标准化，因此其可以用于比较来自不同搜索的比对分数。术语“比特分数”和“相似性分数”可互换使用。比特分数表示比对好的程度；分数越高，比对越好。The bit score S' is derived from the raw alignment score S, which has taken into account the statistical properties of the scoring system used. The bit score is normalized against the scoring system so it can be used to compare alignment scores from different searches. The terms "bit score" and "similarity score" are used interchangeably. The bit score indicates how well the alignment is; the higher the score, the better the alignment.

E值或预期值描述了具有相似分数的序列偶然出现在数据库中的可能性。E值或预期值是预测在数据库检索中偶然出现的具有等同于或优于S的分数的不同比对数量的预测。E值越小，比对越显著。例如，E值为e^-117的比对意味着具有相似分数的序列很不可能只是偶然出现。另外，比对随机氨基酸对的预期分数要求是负的，否则长比对将倾向于具有高分数而不管与比对的区段是否相关。另外，BLAST算法使用适当的取代矩阵、核苷酸或氨基酸，并且对于空位比对，使用空位产生和延伸罚分。例如，BLAST比对和多肽序列的比较通常使用BLOSUM62矩阵、空位存在罚分11以及空位延伸罚分1进行。The E value, or expected value, describes the likelihood that sequences with similar scores will appear in the database by chance. The E value or expected value is the prediction of the number of distinct alignments with a score equal to or better than S by chance in a database search. The smaller the E value, the more significant the alignment. For example, an alignment with an E value of e-¹¹⁷ means that it is very unlikely that sequences with similar scores occurred by chance. Additionally, the expected score requirement for aligning random amino acid pairs is negative, otherwise long alignments will tend to have high scores regardless of whether or not the aligned segment is related. Additionally, the BLAST algorithm uses the appropriate substitution matrix, nucleotide or amino acid, and for gap alignment, gap creation and extension penalties. For example, BLAST alignments and comparisons of polypeptide sequences are typically performed using the BLOSUM62 matrix, a gap presence penalty of 11, and a gap extension penalty of 1.

在一些实施例中，从使用BLOSUM62矩阵、空位存在罚分11以及空位延伸罚分1进行的BLAST分析中报告序列相似性分数。In some embodiments, the sequence similarity score is reported from a BLAST analysis using the BLOSUM62 matrix, a gap presence penalty of 11, and a gap extension penalty of 1.

在一个特定实施例中，本文提供的序列同一性/相似性分数是指利用GAP版本10(GCG，Accelrys，圣地亚哥，加利福尼亚)使用以下参数获得的值：使用GAP权重50和长度权重3以及nwsgapdna.cmp评分矩阵的核苷酸序列的百分比同一性和百分比相似性；使用GAP权重8和长度权重2以及BLOSUM62评分矩阵(Henikoff和Henikoff，《美国科学院院报(PNASUSA)》89:10915-10919,1992)的氨基酸序列的百分比同一性和百分比相似性。GAP利用Needleman和Wunsch的算法(《分子生物学杂志(J Mol Biol.)》48:443-453,1970)来找到两个完整序列的比对，所述比对将匹配数最大化并将空位数最小化。In a specific embodiment, the sequence identity/similarity scores provided herein refer to values obtained using GAP version 10 (GCG, Accelrys, San Diego, CA) using the following parameters: using GAP weight 50 and length weight 3 and nwsgapdna. Percent identity and percent similarity of nucleotide sequences for cmp scoring matrix; using GAP weight 8 and length weight 2 and BLOSUM62 scoring matrix (Henikoff and Henikoff, Proceedings of the National Academy of Sciences (PNASUSA) 89:10915-10919, 1992 ) percent identity and percent similarity of amino acid sequences. GAP utilizes the algorithm of Needleman and Wunsch (J Mol Biol. 48:443-453, 1970) to find an alignment of two complete sequences that maximizes the number of matches and removes gaps numbers are minimized.

在特定实施例中，变体多肽包括可以与参考多肽序列最佳比对的氨基酸序列(参见例如，序列表)，以产生至少约50、60、70、80、90、100、100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、290、300、310、320、330、340、350、360、370、380、390、400、410、420、430、440、450、460、470、480、490、500、510、520、530、540、550、560、570、580、590、600、610、620、630、640、650、660、670、680、690、700、710、720、730、740、750、760、770、780、790、800、810、820、830、840、850、860、870、880、890、900、910、920、930、940、950、960、970、980、990、1000或更多的BLAST比特分数或序列相似性分数(包含其间的所有整数和范围)，其中BLAST比对使用BLOSUM62矩阵、空位存在罚分11以及空位延伸罚分1。In particular embodiments, variant polypeptides include amino acid sequences that can be optimally aligned with a reference polypeptide sequence (see, eg, the Sequence Listing) to yield at least about 50, 60, 70, 80, 90, 100, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 or more BLAST bit scores or sequence similarity scores (including all integers and ranges in between), where BLAST alignments use the BLOSUM62 matrix, a gap presence penalty of 11, and a gap extension penalty of 1.

如上所述，参考多肽可以以各种方式改变，包含氨基酸取代、缺失、截短、添加和插入。用于这种操作的方法是本技术领域公知的。例如，可以通过DNA的突变来制备参考多肽的氨基酸序列变体。用于诱变和核苷酸序列更改的方法是本领域熟知的。参见例如Kunkel(《美国科学院院报(PNAS USA)》82:488-492,1985)；Kunkel等人，(《酶学方法(Methods inEnzymol)》154:367-382,1987)，美国专利第4,873,192号，Watson,J.D.等人(《基因分子生物学(Molecular Biology of the Gene)》第四版，本杰明-卡明斯公司，门洛帕克，加利福尼亚，1987)和本文所引用的参考文献。关于不影响关注蛋白质的生物活性的适当氨基酸取代的指导可以发现于Dayhoff等人，(1978)《蛋白质序列和结构(Atlas of ProteinSequence and Structure)》(美国国家生物医学研究基金会，华盛顿特区)。As noted above, a reference polypeptide can be altered in various ways, including amino acid substitutions, deletions, truncations, additions, and insertions. Methods for such manipulations are well known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutation of DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, eg, Kunkel (PNAS USA 82:488-492, 1985); Kunkel et al. (Methods in Enzymol 154:367-382, 1987), US Pat. No. 4,873,192 No., Watson, J.D. et al. (Molecular Biology of the Gene, Fourth Edition, Benjamin-Cummings, Inc., Menlo Park, Calif., 1987) and references cited herein. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (National Foundation for Biomedical Research, Washington, DC).

用于筛选通过这种修饰制备的组合文库的基因产物的方法，以及用于筛选具有所选性质的基因产物的cDNA文库的方法是本领域已知的。这种方法适用于快速筛选通过参考多肽的组合诱变产生的基因文库。作为一个实例，递归总体诱变(REM)——一种增强文库中功能性突变体频率的技术，可以与筛选试验组合使用以鉴定多肽变体(Arkin和Yourvan，《美国科学院院报》89:7811-7815，1992；Delgrave等人，《蛋白质工程(ProteinEngineering)》6:327-331,1993)。Methods for screening gene products of combinatorial libraries prepared by such modifications, as well as methods for screening cDNA libraries for gene products with selected properties, are known in the art. This method is suitable for rapid screening of gene libraries generated by combinatorial mutagenesis of reference polypeptides. As an example, recursive global mutagenesis (REM), a technique for enhancing the frequency of functional mutants in a library, can be used in combination with screening assays to identify polypeptide variants (Arkin and Yourvan, Proceedings of the National Academy of Sciences 89: 7811-7815, 1992; Delgrave et al., Protein Engineering 6:327-331, 1993).

多肽修饰某些实施例包含包括至少一种“修饰剂”的缀合物，其实例包含但不限于大分子聚合物、蛋白质、肽、多糖和其它化合物。在一些情况下，修饰剂附着于缀合物的ADI组分或缀合物的肿TNF超家族配体组分或两者上。在一些实施例中，修饰剂仅附着于缀合物的ADI组分上，即修饰剂不附着于缀合物的TNF超家族配体(例如，TRAIL)组分上。缀合物和修饰剂可以通过共价键或非共价相互作用连接以形成稳定的缀合物或稳定的组合物以实现期望的效果。在某些实施例中，经过修饰的缀合物保留相应未修饰的缀合物(例如，具有相同或相似序列)的生物活性，并且具有比相应未修饰的缀合物更长的体内半衰期和更低的抗原性。在某些实施例中，已过修饰的缀合物保留相应未修饰的缀合物至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更多的生物活性。通常，经过修饰的缀合物保留足以用于治疗用途的生物活性。Polypeptide Modifications Certain embodiments include conjugates that include at least one "modifying agent," examples of which include, but are not limited to, macromolecular polymers, proteins, peptides, polysaccharides, and other compounds. In some cases, the modifier is attached to the ADI component of the conjugate or the TNF superfamily ligand component of the conjugate, or both. In some embodiments, the modifier is attached only to the ADI component of the conjugate, ie, the modifier is not attached to the TNF superfamily ligand (eg, TRAIL) component of the conjugate. The conjugate and modifier can be linked by covalent bonds or non-covalent interactions to form stable conjugates or stable compositions to achieve the desired effect. In certain embodiments, the modified conjugates retain the biological activity of the corresponding unmodified conjugates (eg, have the same or similar sequence) and have a longer in vivo half-life than the corresponding unmodified conjugates and lower antigenicity. In certain embodiments, the modified conjugate retains at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more biological activity. Generally, the modified conjugates retain sufficient biological activity for therapeutic use.

在一些实施例中，修饰剂是生物相容的聚合物或蛋白质或其片段，并且增加血液中缀合物的半衰期。修饰剂可以化学偶联到缀合物或其组分，或者在适用的情况下，通过融合蛋白表达连接到缀合物或其组分。In some embodiments, the modifier is a biocompatible polymer or protein or fragment thereof, and increases the half-life of the conjugate in blood. Modifiers can be chemically coupled to the conjugate or components thereof, or, where applicable, linked to the conjugate or components thereof by fusion protein expression.

高分子聚合物可以包含非肽高分子聚合物，其在某些实施例中可以具有其自身的生物活性。合适的聚合物包含但不限于多烯醇化合物、聚醚化合物、聚乙烯吡咯烷酮、多聚氨基酸、二乙烯醚和马来酸酐的共聚物、N-(2-羟丙基)-甲基丙烯酰胺、多糖、聚氧乙基多元醇、肝素或其片段、聚烷基乙二醇和其衍生物，聚烷基乙二醇和其衍生物的共聚物、聚(乙烯基乙醚)、a,P-聚[(2-羟乙基)-DL-天冬酰胺]、聚羧酸盐、聚氧乙烯-氧亚甲基、聚丙烯酰吗啉、氨基化合物和氧烯烃的共聚物、聚透明质酸、聚环氧化物、乙二酸和丙二酸的共聚物、聚(1,3-二氧戊环)、乙烯和马来酰肼共聚物、聚唾液酸、环糊精等。在某些实施例中，所述聚合物是聚乙二醇。The high molecular weight polymer may comprise a non-peptidic high molecular weight polymer, which in certain embodiments may have its own biological activity. Suitable polymers include, but are not limited to, polyenol compounds, polyether compounds, polyvinylpyrrolidone, polyamino acids, copolymers of divinyl ether and maleic anhydride, N-(2-hydroxypropyl)-methacrylamide , polysaccharides, polyoxyethyl polyols, heparin or its fragments, polyalkylene glycols and their derivatives, copolymers of polyalkylene glycols and their derivatives, poly(vinyl ether), a,P-polyethylene glycol [(2-hydroxyethyl)-DL-asparagine], polycarboxylate, polyoxyethylene-oxymethylene, polyacryloylmorpholine, copolymer of amino compound and oxyalkene, polyhyaluronic acid, Polyepoxide, copolymer of oxalic acid and malonic acid, poly(1,3-dioxolane), copolymer of ethylene and maleic hydrazide, polysialic acid, cyclodextrin, etc. In certain embodiments, the polymer is polyethylene glycol.

如本文所使用的多烯醇化合物包含但不限于聚乙二醇(包含单甲氧基聚乙二醇、单羟基聚乙二醇)、聚乙烯醇、聚丙烯醇、聚丁烯醇等，以及其衍生物，如脂质。Polyenol compounds as used herein include, but are not limited to, polyethylene glycol (including monomethoxy polyethylene glycol, monohydroxy polyethylene glycol), polyvinyl alcohol, polypropylene alcohol, polybutenol, and the like, and its derivatives, such as lipids.

聚醚化合物包含但不限于聚亚烷基二醇(HO((CH2)_xO)_nH)、聚丙二醇、聚氧化乙烯(HO((CH₂)₂O)_nH)、聚乙烯醇((CH₂CHOH)_n)。Polyether compounds include, but are not limited to, polyalkylene glycol (HO((CH2)_xO )_nH ), polypropylene glycol, polyethylene oxide (HO((_CH2 )_2O )_nH ), polyvinyl alcohol ( (CH₂ CHOH)_n ).

多聚氨基酸包含但不限于一种氨基酸的聚合物或两种或更多种氨基酸的共聚物，例如，聚丙氨酸或聚赖氨酸，或其嵌段共聚物。Polyamino acids include, but are not limited to, polymers of one amino acid or copolymers of two or more amino acids, eg, polyalanine or polylysine, or block copolymers thereof.

多糖包含但不限于葡聚糖和其衍生物，例如硫酸葡聚糖、纤维素和其衍生物(包含甲基纤维素和羧甲基纤维素)、淀粉和其衍生物、聚蔗糖等。Polysaccharides include, but are not limited to, dextran and derivatives thereof, such as dextran sulfate, cellulose and derivatives thereof (including methylcellulose and carboxymethylcellulose), starch and derivatives thereof, polysucrose, and the like.

在特定实施例中，修饰剂是PEG分子。“聚乙二醇”或“PEG”是指支链或直链的环氧乙烷和水的缩聚物的混合物，由通式H(OCH₂CH₂)_nOH表示，其中n至少为4。在一些情况下，PEG附着于缀合物的ADI组分或缀合物的肿TNF超家族配体组分或两者上。在一些实施例中，PEG仅附着于缀合物的ADI组分上，即PEG不附着于缀合物的TNF超家族配体(例如，TRAIL)组分上。In certain embodiments, the modifier is a PEG molecule. "Polyethylene glycol" or "PEG" refers to a mixture of branched or linear polycondensates of ethylene oxide and water, represented by the general formula H(_OCH2CH2) nOH, wherein_n is at least 4. In some cases, the PEG is attached to the ADI component of the conjugate or the TNF superfamily ligand component of the conjugate, or both. In some embodiments, the PEG is attached only to the ADI component of the conjugate, ie, the PEG is not attached to the TNF superfamily ligand (eg, TRAIL) component of the conjugate.

“聚乙二醇”或“PEG”与数字后缀组合使用以表示其近似重均分子量。例如，PEG5,000是指总重均分子量为约5,000的PEG；PEG12,000是指总重均分子量为约12,000的PEG；以及PEG20,000是指总重均分子量为约20,000的PEG。"Polyethylene glycol" or "PEG" is used in combination with a numerical suffix to denote its approximate weight average molecular weight. For example, PEG 5,000 refers to PEG having a total weight average molecular weight of about 5,000; PEG 12,000 refers to PEG having a total weight average molecular weight of about 12,000; and PEG 20,000 refers to PEG having a total weight average molecular weight of about 20,000.

在一些实施例中，PEG的总重均分子量为约1,000到约50,000；约3,000到约40,000；约5,000到约30,000；约8,000到约30,000；约11,000到约30,000；约12,000到约28,000；约16,000到约24,000；约18,000到约22,000；或约19,000到约21,000。在一些实施例中，PEG的总重均分子量为约1,000到约50,000；约3,000到约30,000；约3,000到约20,000；约4,000到约12,000；约4,000到约10,000；约4,000到约8,000；约4,000到约6,000；或约5,000。在具体实施例中，PEG的总重均分子量为约20,000。通常，分子量为30,000或更多的PEG难以溶解，并且配制产品的产率可能降低。PEG可以是支链或直链。PEG可以是支链或直链，并且在某些实施例中是直链。具有本文所述的分子量的PEG可以与缀合物或其组分结合使用，并且任选地，与生物相容性连接子结合使用。In some embodiments, the PEG has a total weight average molecular weight of about 1,000 to about 50,000; about 3,000 to about 40,000; about 5,000 to about 30,000; about 8,000 to about 30,000; 16,000 to about 24,000; about 18,000 to about 22,000; or about 19,000 to about 21,000. In some embodiments, the PEG has a total weight average molecular weight of about 1,000 to about 50,000; about 3,000 to about 30,000; about 3,000 to about 20,000; about 4,000 to about 12,000; 4,000 to about 6,000; or about 5,000. In specific embodiments, the PEG has a total weight average molecular weight of about 20,000. Typically, PEGs with molecular weights of 30,000 or more are difficult to dissolve, and the yield of formulated products may be reduced. PEG can be branched or straight chain. PEG can be branched or linear, and in certain embodiments linear. PEGs having the molecular weights described herein can be used in conjunction with conjugates or components thereof, and optionally, in conjunction with biocompatible linkers.

某些实施例使用一个或多个硫醇、巯基或半胱氨酸反应性PEG。在一些实施例中，一个或多个硫醇、巯基或半胱氨酸反应性PEG附着于一个或多个天然存在的半胱氨酸残基、一个或多个引入的半胱氨酸残基(例如，用一个或多个半胱氨酸残基取代一个或多个野生型残基、插入一个或多个半胱氨酸残基)或其任何组合(参见例如，Doherty等人，《生物共轭化学(Bioconjug Chem.)》16:1291-98,2005)。在具体实施例中，缀合物的ADI组分具有K192C和/或K287C取代中的一个或两个，用于附着到一个或多个半胱氨酸反应性PEG。在某些实施例中，缀合物的一个或多个野生型半胱氨酸残基可以被另一种氨基酸取代，以防止PEG聚合物与野生型半胱氨酸的连接，例如，以防止一个或多个PEG破坏其它期望的生物活性。一些实施例采用一种或多种非天然半胱氨酸衍生物(例如，高半胱氨酸)，而非半胱氨酸。Certain embodiments use one or more thiol, sulfhydryl, or cysteine-reactive PEGs. In some embodiments, one or more thiol, sulfhydryl or cysteine reactive PEGs are attached to one or more naturally occurring cysteine residues, one or more introduced cysteine residues (eg, substitution of one or more cysteine residues for one or more wild-type residues, insertion of one or more cysteine residues) or any combination thereof (see, eg, Doherty et al., Biological Bioconjug Chem. 16:1291-98, 2005). In specific embodiments, the ADI component of the conjugate has one or both of the K192C and/or K287C substitutions for attachment to one or more cysteine-reactive PEGs. In certain embodiments, one or more wild-type cysteine residues of the conjugate can be substituted with another amino acid to prevent attachment of the PEG polymer to the wild-type cysteine, eg, to prevent One or more PEGs disrupt other desired biological activities. Some embodiments employ one or more non-natural cysteine derivatives (eg, homocysteine) instead of cysteine.

硫醇、巯基或半胱氨酸反应性PEG的非限制性实例包含甲氧基PEG马来酰亚胺(M-PEG-MAL)(例如，MW 2000、MW 5000、MW 10000、MW 20000、MW 30000、MW 40000)。M-PEG-MAL与蛋白质和肽中的半胱氨酸侧链上的硫醇基团反应，以产生稳定的3-硫代琥珀酰亚胺基醚键。此反应具有高选择性，可在约pH 5.0-6.5的温和条件下，在其它官能团存在下进行。因此，在某些实施例中，缀合物或其组分缀合到本文所述的硫醇、巯基或半胱氨酸反应性PEG分子的任何一种或多种。Non-limiting examples of thiol, sulfhydryl or cysteine reactive PEGs include methoxyPEG maleimide (M-PEG-MAL) (eg, MW 2000, MW 5000, MW 10000, MW 20000, MW 30000, MW 40000). M-PEG-MAL reacts with thiol groups on cysteine side chains in proteins and peptides to generate stable 3-thiosuccinimidyl ether linkages. This reaction is highly selective and can be carried out under mild conditions at about pH 5.0-6.5 in the presence of other functional groups. Thus, in certain embodiments, the conjugates or components thereof are conjugated to any one or more of the thiol, sulfhydryl or cysteine reactive PEG molecules described herein.

缀合物或其组分可以在有连接子或没有连接子的情况下共价键合到修饰剂(如PEG)。在一些情况下，缀合物或其组分可以直接(即，在没有连接子的情况下)偶联到修饰剂(如PEG)，例如，通过氨基、巯基、羟基、羧基或其它基团。The conjugate or its components can be covalently bonded to a modifier (eg, PEG) with or without a linker. In some cases, the conjugate or its components can be coupled directly (ie, without a linker) to a modifier (eg, PEG), eg, through an amino, sulfhydryl, hydroxyl, carboxyl, or other group.

用于将缀合物或其组分共价附着到修饰剂(例如，PEG)的连接子可以是任何生物相容性连接子。“生物相容的”表示化合物或基团是无毒的并且可以在体外或体内使用而不会引起损伤、疾病、病症或死亡。修饰剂(如PEG)可以例如通过醚键、硫醇键、酰胺键或其它键键合到连接子。The linker used to covalently attach the conjugate or its components to the modifying agent (eg, PEG) can be any biocompatible linker. "Biocompatible" means that the compound or group is nontoxic and can be used in vitro or in vivo without causing injury, disease, disorder, or death. Modifiers, such as PEG, can be bonded to the linker, for example, through ether, thiol, amide, or other linkages.

在一些实施例中，合适的连接子可以具有例如约1-100个原子、1-80个原子、1-60个原子、1-40个原子、1-30个原子、1-20个原子，或1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个原子的总链长，其中链中的原子包括C、S、N、P、和/或O。在一些情况下，连接子基团包含例如琥珀酰基、酰胺基、酰亚胺基、氨基甲酸酯基、酯基、环氧基、羧基、羟基、碳水化合物、酪氨酸基、半胱氨酸基、组氨酸基、亚甲基、以及其组合。稳定连接子的特定实例包含琥珀酰亚胺基、丙酸、羧甲基连接、醚、氨基甲酸酯、酰胺、胺、脲、酰亚胺、脂族C-C键和硫醚。在某些实施例中，生物相容性连接子是琥珀酰亚胺基琥珀酸酯(SS)基团。In some embodiments, suitable linkers may have, for example, about 1-100 atoms, 1-80 atoms, 1-60 atoms, 1-40 atoms, 1-30 atoms, 1-20 atoms, or a total chain length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 atoms, where in the chain The atoms include C, S, N, P, and/or O. In some cases, the linker group includes, for example, a succinyl group, an amide group, an imide group, a carbamate group, an ester group, an epoxy group, a carboxyl group, a hydroxyl group, a carbohydrate, a tyrosine group, a cysteine group Acid groups, histidine groups, methylene groups, and combinations thereof. Specific examples of stable linkers include succinimidyl, propionic acid, carboxymethyl linkages, ethers, carbamates, amides, amines, ureas, imides, aliphatic C-C bonds, and thioethers. In certain embodiments, the biocompatible linker is a succinimidyl succinate (SS) group.

其它合适的连接子包含氧羰基咪唑基(包含，例如，羰基咪唑(CDI))、硝基苯基(包含，例如，硝基苯基碳酸酯(NCP)或三氯苯基碳酸酯(TCP))、甲苯磺酸酯基、醛基、异氰酸酯基、乙烯基砜基或伯胺。在某些实施例中，连接子衍生自SS、SPA、SCM或NHS；在某些实施例中，使用SS、SPA或NHS，并且在一些实施例中，使用SS或SPA。因此，在某些实施例中，潜在的连接子可以由甲氧基-PEG琥珀酰亚胺基琥珀酸酯(SS)、甲氧基-PEG琥珀酰亚胺基戊二酸酯(SG)、甲氧基-PEG琥珀酰亚胺基碳酸酯(SC)、甲氧基-PEG琥珀酰亚胺基羧甲基酯(SCM)、甲氧基-PEG2 N-羟基琥珀酰亚胺基(NHS)、甲氧基-PEG琥珀酰亚胺基丁酸酯(SBA)、甲氧基-PEG琥珀酰亚胺基丙酸酯(SPA)、甲氧基-PEG琥珀酰亚胺基戊二酰胺和/或甲氧基-PEG琥珀酰亚胺基琥珀酰亚胺形成。Other suitable linkers include oxycarbonyl imidazolyl (including, eg, carbonylimidazole (CDI)), nitrophenyl (including, eg, nitrophenyl carbonate (NCP) or trichlorophenyl carbonate (TCP) ), tosylate group, aldehyde group, isocyanate group, vinylsulfone group or primary amine. In certain embodiments, the linker is derived from SS, SPA, SCM, or NHS; in certain embodiments, SS, SPA, or NHS is used, and in some embodiments, SS or SPA is used. Thus, in certain embodiments, potential linkers may be composed of methoxy-PEG succinimidyl succinate (SS), methoxy-PEG succinimidyl glutarate (SG), Methoxy-PEG Succinimidyl Carbonate (SC), Methoxy-PEG Succinimidyl Carboxymethyl Ester (SCM), Methoxy-PEG2 N-Hydroxysuccinimidyl (NHS) , Methoxy-PEG Succinimidyl Butyrate (SBA), Methoxy-PEG Succinimidyl Propionate (SPA), Methoxy-PEG Succinimidyl Glutaramide and/ or methoxy-PEG succinimidyl succinimide formation.

连接子的附加实例包含但不限于以下一种或多种：—O—、—NH—、—S—、—C(O)—、C(O)—NH、NH—C(O)—NH、O—C(O)—NH、—C(S)—、—CH2—、—CH2—CH2—、—CH2—CH2—CH2—、—CH2—CH2—CH2—CH2—、—O—CH2—、—CH2—O—、—O—CH2—CH2—、—CH2—O—CH2—、—CH2—CH2—O—、—O—CH2—CH2—CH2—、—CH2—O—CH2—CH2—、—CH2—CH2—O—CH2—、—CH2—CH2—CH2—O—、—O—CH2—CH2—CH2—CH2—、—CH2—O—CH2—CH2—CH2—、—CH2—CH2—O—CH2—CH2—、—CH2—CH2—CH2—O—CH2—、—CH2—CH2—CH2—CH2—O—、—C(O)—NH—CH2—、—C(O)—NH—CH2—CH2—、—CH2—C(O)—NH—CH2—、—CH2—CH2—C(O)—NH—、—C(O)—NH—CH2—CH2—CH2—、—CH2—C(O)—NH—CH2—CH2—、—CH2—CH2—C(O)—NH—CH2—、—CH2—CH2—CH2—C(O)—NH—、—C(O)—NH—CH2—CH2—CH2—CH2—、—CH2—C(O)—NH—CH2—CH2—CH2—、—CH2—CH2—C(O)—NH—CH2—CH2—、—CH2—CH2—CH2—C(O)—NH—CH2—、—CH2—CH2—CH2—C(O)—NH—CH2—CH2—、—CH2—CH2—CH2—CH2—C(O)—NH—、—NH—C(O)—CH2—、—CH2—NH—C(O)—CH2—、—CH2—CH2—NH—C(O)—CH2—、—NH—C(O)—CH2—CH2—、—CH2—NH—C(O)—CH2—CH2、—CH2—CH2—NH—C(O)—CH2—CH2、—C(O)—NH—CH2—、—C(O)—NH—CH2—CH2—、—O—C(O)—NH—CH2—、—O—C(O)—NH—CH2—CH2—、—NH—CH2—、—NH—CH2—CH2—、—CH2—NH—CH2—、—CH2—CH2—NH—CH2—、—C(O)—CH2—、—C(O)—CH2—CH2—、—CH2—C(O)—CH2—、—CH2—CH2—C(O)—CH2—、—CH2—CH2—C(O)—CH2—CH2—、—CH2—CH2—C(O)—、—CH2—CH2—CH2—C(O)—NH—CH2—CH2—NH—、—CH2—CH2—CH2—C(O)—NH—CH2—CH2—NH—C(O)—、—CH2—CH2—CH2—C(O)—NH—CH2—CH2—NH—C(O)—CH2—、二价环烷基、—N(R6)—，R6是H或选自由烷基、取代的烷基、烯基、取代的烯基、炔基、取代的炔基、芳基和取代的芳基组成的基团的有机基。另外，本文所述的任何连接子部分可以进一步包含环氧乙烷低聚物链，其包括1到20个环氧乙烷单体单元[即，-(CH₂CH₂O)_1-20-]。也就是说，环氧乙烷低聚物链可以在连接子之前或之后出现，并且任选地出现在包括两个或更多个原子的连接子部分的任何两个原子之间。并且，如果低聚物与聚合物区段相邻并且仅代表聚合物区段的延伸，则低聚物链将不被认为是连接子部分的一部分。Additional examples of linkers include, but are not limited to, one or more of the following: —O—, —NH—, —S—, —C(O)—, C(O)—NH, NH—C(O)—NH , O—C(O)—NH,—C(S)—,—CH2—,—CH2—CH2—,—CH2—CH2—CH2—,—CH2—CH2—CH2—CH2—,—O—CH2— ,—CH2—O—,—O—CH2—CH2—,—CH2—O—CH2—,—CH2—CH2—O—,—O—CH2—CH2—CH2—,—CH2—O—CH2—CH2— ,—CH2—CH2—O—CH2—,—CH2—CH2—CH2—O—,—O—CH2—CH2—CH2—CH2—,—CH2—O—CH2—CH2—CH2—,—CH2—CH2— O—CH2—CH2—,—CH2—CH2—CH2—O—CH2—,—CH2—CH2—CH2—CH2—O—,—C(O)—NH—CH2—,—C(O)—NH— CH2—CH2—,—CH2—C(O)—NH—CH2—,—CH2—CH2—C(O)—NH—,—C(O)—NH—CH2—CH2—CH2—,—CH2—C (O)—NH—CH2—CH2—,—CH2—CH2—C(O)—NH—CH2—,—CH2—CH2—CH2—C(O)—NH—,—C(O)—NH—CH2 —CH2—CH2—CH2—,—CH2—C(O)—NH—CH2—CH2—CH2—,—CH2—CH2—C(O)—NH—CH2—CH2—,—CH2—CH2—CH2—C (O)—NH—CH2—,—CH2—CH2—CH2—C(O)—NH—CH2—CH2—,—CH2—CH2—CH2—CH2—C(O)—NH—,—NH—C( O)—CH2—,—CH2—NH—C(O)—CH2—,—CH2—CH2—NH—C(O)—CH2—,—NH—C(O)—CH2—CH2—,—CH2— NH—C(O)—CH2—CH2,—CH2—CH2—NH—C(O)—CH2—CH2,—C(O)—NH—CH2—,—C(O)—NH—CH2—CH2— ,—O—C(O)—NH—CH2—,—O—C(O)—NH—CH2—CH2—,—NH—CH2—,—NH—CH2—CH2—,—CH2—NH—CH2— ,—CH2—CH2—NH—CH2—,—C(O)—CH2—,—C(O)—CH2—CH2—,—CH2—C(O)—CH2—,—CH2—CH2—C(O )—CH2—,—CH2—CH2—C(O)—CH2—CH2—,—CH2—CH2—C(O)—,—CH2—C H2—CH2—C(O)—NH—CH2—CH2—NH—,—CH2—CH2—CH2—C(O)—NH—CH2—CH2—NH—C(O)—,—CH2—CH2—CH2 -C(O)-NH-CH2-CH2-NH-C(O)-CH2-, divalent cycloalkyl, -N(R6)-, R6 is H or selected from alkyl, substituted alkyl, alkene The organic group of the group consisting of alkynyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl and substituted aryl. Additionally, any linker moiety described herein may further comprise an ethylene oxide oligomer chain comprising 1 to₂₀ ethylene oxide monomer units [ie, -(_CH2CH2O )_1-20- ]. That is, the ethylene oxide oligomer chain can occur before or after the linker, and optionally between any two atoms of a linker moiety that includes two or more atoms. Also, if the oligomer is adjacent to the polymer segment and represents only an extension of the polymer segment, the oligomer chain would not be considered part of the linker moiety.

下表P1中描述了具体的示例性PEG分子和连接子。Specific exemplary PEG molecules and linkers are described in Table P1 below.

在某些实施例中，缀合物或其组分包括一种或多种如本文(例如，在表P1中)所描述的PEG分子和/或连接子。In certain embodiments, the conjugates or components thereof include one or more PEG molecules and/or linkers as described herein (eg, in Table P1).

1到约30个PEG分子可以共价键合到缀合物或其组分。在某些实施例中，缀合物或其组分用一个PEG分子修饰(即，包括)。在一些实施例中，缀合物或其组分用多于一个PEG分子修饰。在特定实施例中，缀合物或其组分用约1到约10、或约7到约15个PEG分子，或约2到约8或约9到约12个PEG分子修饰。在一些实施例中，缀合物或其组分用约2、3、4、5、6、7、8、9、10、11、12、13、14或15个PEG分子修饰。在具体实施例中，缀合物或其组分用每缀合物4.5-5.5个PEG分子修饰。在一些实施例中，缀合物或其组分用5±1.5个PEG分子修饰。From 1 to about 30 PEG molecules can be covalently bonded to the conjugate or a component thereof. In certain embodiments, the conjugate or component thereof is modified (ie, includes) with one PEG molecule. In some embodiments, the conjugate or components thereof are modified with more than one PEG molecule. In particular embodiments, the conjugate or component thereof is modified with about 1 to about 10, or about 7 to about 15 PEG molecules, or about 2 to about 8 or about 9 to about 12 PEG molecules. In some embodiments, the conjugate or component thereof is modified with about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 PEG molecules. In specific embodiments, the conjugate or components thereof are modified with 4.5-5.5 PEG molecules per conjugate. In some embodiments, the conjugate or component thereof is modified with 5±1.5 PEG molecules.

在某些实施例中，缀合物或其组分中约15％到约70％的伯氨基用PEG修饰，在一些实施例中精氨酸脱亚胺酶中约20％到约65％、约25％到约60％，或在某些实施例中约30％到约55％、或45％到约50％，或在一些实施例中约50％的伯氨基用PEG修饰。In certain embodiments, about 15% to about 70% of the primary amino groups in the conjugate or components thereof are modified with PEG, in some embodiments about 20% to about 65% in the arginine deiminase, About 25% to about 60%, or in certain embodiments about 30% to about 55%, or 45% to about 50%, or in some embodiments about 50% of the primary amino groups are modified with PEG.

附着到缀合物的PEG可以与SS-PEG、SPA-PEG和SC-PEG一样，是直链，或与PEG2-NHS一样，可以使用PEG的支链。The PEG attached to the conjugate can be straight chain like SS-PEG, SPA-PEG and SC-PEG, or branched chain of PEG can be used like PEG2-NHS.

在一些实施例中，例如，如上所述，氨基酸取代采用非天然氨基酸缀合到PEG或其它修饰剂(参见例如，de Graaf等人，《生物缀合化学(Bioconjug Chem.)》20:1281-95,2009)。因此，某些实施例包含通过一个或多个非天然氨基酸缀合到一个或多个PEG的缀合物或其组分。在一些实施例中，非天然氨基酸包括具有官能团的侧链，所述官能团选自以下组成的组：烷基、芳基、芳基卤、乙烯基卤、烷基卤、乙酰基、酮、氮杂环丙烷、腈、硝基、卤化物、酰基、酮基、叠氮基、羟基、肼、氰基、卤素、酰肼、烯基、炔基、醚、硫醚、环氧化物、砜、硼酸、硼酸酯、硼烷、苯硼酸、硫醇、硒基、磺酰基，硼酸酯、硼酸盐，膦基、膦羧基、磷化氢、杂环基、氮苯基、萘基、苯甲酮、受限环如环辛炔、硫酯、烯酮、亚胺、醛、酯、硫代酸、羟胺、氨基、羧酸、α-酮羧酸、α或β不饱和酸和酰胺、乙醛酰胺和有机硅烷基团。在一些实施例中，非天然氨基酸选自以下组成的组：对乙酰基-L-苯丙氨酸、O-甲基-L-酪氨酸、L-3-(2-萘基)丙氨酸、3-甲基-苯丙氨酸、O-4-烯丙基-L-酪氨酸、高半胱氨酸、4-丙基-L-酪氨酸、三-O-乙酰基-GlcNAcβ-丝氨酸、β-O-GlcNAc-L-丝氨酸、三-O-乙酰基-GalNAc-α-苏氨酸、α-GalNAc-L-苏氨酸、左旋多巴、氟化苯丙氨酸、异丙基-L-苯丙氨酸、对叠氮-L-苯丙氨酸、对酰基-L-苯丙氨酸，对苯甲酰基-L-苯丙氨酸、L-磷酸丝氨酸、膦丝氨酸、膦酰酪氨酸、对碘苯丙氨酸、对溴苯丙氨酸、对氨基-L-苯丙氨酸、以及异丙基-L-苯丙氨酸。In some embodiments, eg, as described above, amino acid substitutions employ unnatural amino acids conjugated to PEG or other modifiers (see eg, de Graaf et al., Bioconjug Chem. 20:1281- 95, 2009). Accordingly, certain embodiments comprise conjugates or components thereof conjugated to one or more PEGs via one or more unnatural amino acids. In some embodiments, the unnatural amino acid includes side chains having functional groups selected from the group consisting of: alkyl, aryl, aryl halide, vinyl halide, alkyl halide, acetyl, ketone, nitrogen Heterocyclopropane, nitrile, nitro, halide, acyl, keto, azide, hydroxyl, hydrazine, cyano, halogen, hydrazide, alkenyl, alkynyl, ether, thioether, epoxide, sulfone, Boric acid, boronate, borane, phenylboronic acid, thiol, selenoyl, sulfonyl, borate, borate, phosphine, phosphine, phosphine, heterocyclyl, nitrogen phenyl, naphthyl, Benzophenones, restricted rings such as cyclooctynes, thioesters, enones, imines, aldehydes, esters, thioacids, hydroxylamines, amino acids, carboxylic acids, alpha-ketocarboxylic acids, alpha or beta unsaturated acids and amides , glyoxylamide and organosilane groups. In some embodiments, the unnatural amino acid is selected from the group consisting of p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine acid, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, homocysteine, 4-propyl-L-tyrosine, tri-O-acetyl- GlcNAc β-serine, β-O-GlcNAc-L-serine, tri-O-acetyl-GalNAc-α-threonine, α-GalNAc-L-threonine, L-dopa, fluorinated phenylalanine, Isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphine Serine, phosphonotyrosine, p-iodophenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, and isopropyl-L-phenylalanine.

多核苷酸、表达载体和宿主细胞某些实施例涉及编码缀合物(例如，如本文所述的融合多肽)的多核苷酸。还包含单独地或与编码本文所述的任何一种或多种单独TNF超家族配体或三聚体多肽的多核苷酸组合地编码本文所述的任何一种或多种单独ADI或六聚体多肽的多核苷酸。因此，某些实施例包含编码表A1中的任何一种或多种单独ADI多肽、表T1或表T2中的任何一种或多种单独TNF超家族配体或本文所述的融合多肽的多核苷酸，例如，包括表A1中的融合到表T1或表T2中任何一种或多种TNF超家族配体的任何一种或多种ADI多肽的融合多肽。Polynucleotides, Expression Vectors and Host Cells Certain embodiments relate to polynucleotides encoding conjugates (eg, fusion polypeptides as described herein). Also included are polynucleotides encoding any one or more of the individual ADIs or hexamers described herein, alone or in combination with a polynucleotide encoding any one or more of the individual TNF superfamily ligand or trimer polypeptides described herein A polynucleotide of a body polypeptide. Thus, certain embodiments comprise multinucleus encoding any one or more individual ADI polypeptides in Table A1, any one or more individual TNF superfamily ligands in Table T1 or Table T2, or fusion polypeptides described herein The nucleotides, for example, include fusion polypeptides of any one or more of the ADI polypeptides in Table A1 fused to any one or more of the TNF superfamily ligands in Table T1 or Table T2.

在其它用途中，这些和相关实施例可以用于在宿主细胞中重组产生融合多肽或其单独组分(ADI、TNF超家族配体、六聚体多肽、三聚体多肽)。本领域的普通技术人员应理解，由于遗传密码的简并性，存在许多编码本文所述的多肽的核苷酸序列。这些多核苷酸中的一些与任何天然基因的核苷酸序列可以具有最小的同源性。尽管如此，具体设想了由于密码子使用的差异而变化的多核苷酸，例如针对人类、酵母或细菌密码子选择而优化的多核苷酸。Among other uses, these and related examples can be used to recombinantly produce fusion polypeptides or individual components thereof (ADIs, TNF superfamily ligands, hexameric polypeptides, trimeric polypeptides) in host cells. One of ordinary skill in the art will appreciate that, due to the degeneracy of the genetic code, there are many nucleotide sequences encoding the polypeptides described herein. Some of these polynucleotides may have minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated, eg, polynucleotides optimized for human, yeast or bacterial codon usage.

如本领域技术人员认识到的，多核苷酸可以是单链(编码或反义)或双链的，并且可以是DNA(基因组、cDNA或合成的)或RNA分子。另外的编码或非编码序列可以但不必存在于本公开的多核苷酸内，并且多核苷酸可以但不必连接到其它分子和/或支持材料。As recognized by those skilled in the art, polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be DNA (genomic, cDNA or synthetic) or RNA molecules. Additional coding or non-coding sequences may but need not be present within the polynucleotides of the present disclosure, and the polynucleotides may but need not be linked to other molecules and/or support materials.

多核苷酸可以包括天然序列(即，编码融合多肽或其组分的内源序列)或可以包括变体或此类序列的生物功能等同物。多核苷酸变体可以含有一个或多个如本文所述的取代、添加、删除和/或插入，优选地使得变体多肽的活性相对于未修饰的多肽基本上未降低。Polynucleotides may include native sequences (ie, endogenous sequences encoding fusion polypeptides or components thereof) or may include variants or biologically functional equivalents of such sequences. Polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions as described herein, preferably such that the activity of the variant polypeptide is not substantially reduced relative to the unmodified polypeptide.

另外的编码或非编码序列可以但不必存在于多核苷酸内，并且多核苷酸可以但不必连接到其它分子和/或支持材料。因此，无论编码序列本身的长度如何，多核苷酸都可以与其它DNA或RNA序列，如启动子、聚腺苷酸化信号、另外的限制酶位点、多克隆位点、其它编码区段等组合，使得它们的总长度可以改变很大。Additional coding or non-coding sequences may, but need not, be present within the polynucleotide, and the polynucleotide may but need not be linked to other molecules and/or support materials. Thus, regardless of the length of the coding sequence itself, polynucleotides can be combined with other DNA or RNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc. , so that their total length can vary greatly.

多核苷酸序列还可以是混合基因组cDNA、RNA的序列以及合成来源的序列。例如，编码前导肽的基因组或cDNA序列可以加入到编码多肽的基因组或cDNA序列，之后可以根据熟知的方法步骤通过插入编码用于同源重组的期望的氨基酸序列的合成寡核苷酸或优选地使用合适的寡核苷酸通过PCR产生期望的序列而在位点处修饰DNA或RNA序列。在一些实施例中，信号序列可以包含在编码序列之前。这个序列将信号肽N端编码到编码序列，所述编码序列与宿主细胞连通以将多肽导向细胞表面或将多肽分泌到培养基中。通常，在蛋白质离开细胞之前，信号肽被宿主细胞修剪掉(clipped off)。可以在原核生物和真核生物中的各种蛋白质中发现信号肽。The polynucleotide sequence may also be of mixed genomic cDNA, RNA, and synthetic origin. For example, a genomic or cDNA sequence encoding a leader peptide can be added to a genomic or cDNA sequence encoding a polypeptide, followed by insertion of synthetic oligonucleotides encoding the desired amino acid sequence for homologous recombination according to well-known method procedures or preferably The DNA or RNA sequence is modified at the site using appropriate oligonucleotides to generate the desired sequence by PCR. In some embodiments, the signal sequence may be included before the coding sequence. This sequence encodes the N-terminus of the signal peptide to a coding sequence that communicates with the host cell to direct the polypeptide to the cell surface or to secrete the polypeptide into the culture medium. Typically, the signal peptide is clipped off by the host cell before the protein leaves the cell. Signal peptides can be found in various proteins in prokaryotes and eukaryotes.

一种或多种多核苷酸可以编码本文所述的ADI、TNF超家族配体、六聚体、三聚体和/或融合多肽。此外，可以出于各种原因操纵多核苷酸序列。实例包含但不限于掺入用于增强各种生物体中多核苷酸的表达的优选密码子(一般参见Nakamura等人，《核酸研究(Nucl Acids Res.)》28:292,2000)。另外，可以掺入沉默突变以引入或消除限制性位点，降低CpG二核苷酸基序的密度(参见，例如，Kameda等人，《生物化学生物物理研究通讯(Biochem.Biophys.Res.Commun.)》349:1269-1277,2006)，或降低单链序列形成茎环结构的能力：(参见，例如，Zuker M.，《核酸研究(Nucl Acids Res.)》31:3406-3415,2003)。另外，在起始密码子处可以通过包含Kozak共有序列(即，(a/g)cc(a/g)ccATGg)(SEQ ID NO:95)，进一步优化哺乳动物表达。用于此目的的Kozak共有序列是本领域已知的(Mantyh等人，《美国国家科学院院刊(PNAS)》92:2662-2666,1995；Mantyh等人，《蛋白质表达与纯化(Prot.Exp.&Purif.)》6:124,1995)。One or more polynucleotides may encode ADIs, TNF superfamily ligands, hexamers, trimers and/or fusion polypeptides described herein. Furthermore, polynucleotide sequences can be manipulated for a variety of reasons. Examples include, but are not limited to, the incorporation of preferred codons for enhancing expression of polynucleotides in various organisms (see generally Nakamura et al., Nucl Acids Res. 28:292, 2000). Additionally, silent mutations can be incorporated to introduce or eliminate restriction sites, reducing the density of CpG dinucleotide motifs (see, eg, Kameda et al., Biochem. Biophys. Res. Commun .)" 349:1269-1277, 2006), or reduce the ability of single-stranded sequences to form stem-loop structures: (see, e.g., Zuker M., Nucl Acids Res. 31:3406-3415, 2003 ). Additionally, mammalian expression can be further optimized by including a Kozak consensus sequence (ie, (a/g)cc(a/g)ccATGg) (SEQ ID NO: 95) at the start codon. Kozak consensus sequences for this purpose are known in the art (Mantyh et al., Proceedings of the National Academy of Sciences (PNAS) 92:2662-2666, 1995; Mantyh et al., Protein Expression and Purification (Prot. Exp. . & Purif.)” 6:124, 1995).

还包含包括多核苷酸的表达载体，以及包括多核苷酸和/或表达载体的宿主细胞。多肽和缀合物，例如融合多肽，可以通过众所周知的技术在合适的宿主细胞中通过表达编码多肽的DNA或RNA序列来产生。术语“宿主细胞”用于指在其中已经引入或能够引入编码一种或多种本文所述多肽的核酸序列的细胞，并且所述细胞进一步表达或能够表达所关注多肽，如编码任何本文所述多肽的多核苷酸。所述术语包含亲本细胞的子代，无论所述子代是否在形态上或遗传构成上与原始亲本相同，只要存在所选基因即可。可以选择宿主细胞用于某些特征，例如，表达甲酰甘氨酸生成酶(FGE)以将硫酸酯酶基序内的半胱氨酸或丝氨酸残基转化为甲酰甘氨酸(FGly)残基，或表达可以将非天然氨基酸，包括具有叠氮侧链、炔侧链或其它期望侧链的非天然氨基酸并入多肽的一种或多种氨酰基tRNA合成酶，以促进化学缀合或修饰。Also included are expression vectors comprising polynucleotides, and host cells comprising polynucleotides and/or expression vectors. Polypeptides and conjugates, such as fusion polypeptides, can be produced by well-known techniques in suitable host cells by expressing DNA or RNA sequences encoding the polypeptides. The term "host cell" is used to refer to a cell into which a nucleic acid sequence encoding one or more of the polypeptides described herein has been introduced or is capable of being introduced, and the cell further expresses or is capable of expressing a polypeptide of interest, such as encoding any of the polypeptides described herein. Polynucleotides of polypeptides. The term includes the progeny of the parent cell, whether or not the progeny is morphologically or genetically identical to the original parent, so long as the selected gene is present. Host cells can be selected for certain features, for example, expressing a formylglycine-generating enzyme (FGE) to convert cysteine or serine residues within a sulfatase motif to formylglycine (FGly) residues, or Expression of one or more aminoacyl tRNA synthetases that can incorporate unnatural amino acids, including unnatural amino acids with azide side chains, alkyne side chains, or other desired side chains, into polypeptides to facilitate chemical conjugation or modification.

在一些情况下，多核苷酸或表达载体包括另外的非编码序列。例如，表达载体中存在的“控制元件”或“调节序列”是载体的非翻译区，包含增强子、启动子，5'和3'非翻译区，其与宿主细胞蛋白相互作用以进行转录和翻译。这些元件的强度和特异性可能不同。根据所利用的载体系统和宿主，可以使用任何数量的适合的转录和翻译元件，包含组成型和诱导型启动子。例如，当在细菌系统中克隆时，可以使用诱导型启动子，如PBLUESCRIPT噬菌粒(Stratagene，加利福利亚州拉荷亚市)或PSPORT1质粒(Gibco BRL，马里兰州盖瑟斯堡市)等的杂合lacZ启动子。在哺乳动物细胞系统中，通常优选来自哺乳动物基因或哺乳动物病毒的启动子。如果需要产生含有编码多肽的序列的多个拷贝的细胞系，则基于SV40或EBV的载体可以有利地与适当的可选标记一起使用。In some cases, the polynucleotide or expression vector includes additional non-coding sequences. For example, "control elements" or "regulatory sequences" present in an expression vector are the untranslated regions of the vector, comprising enhancers, promoters, 5' and 3' untranslated regions, which interact with host cell proteins for transcription and translate. The strength and specificity of these elements may vary. Depending on the vector system and host utilized, any number of suitable transcriptional and translational elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the PBLUESCRIPT phagemid (Stratagene, La Jolla, CA) or the PSPORT1 plasmid (Gibco BRL, Gaithersburg, MD) can be used The hybrid lacZ promoter of et al. In mammalian cell systems, promoters from mammalian genes or mammalian viruses are generally preferred. If it is desired to generate cell lines containing multiple copies of the sequence encoding the polypeptide, SV40 or EBV-based vectors can be advantageously used with an appropriate selectable marker.

各种表达载体/宿主系统是已知的，并且可以用于含有和表达多核苷酸序列。这些包含但不限于微生物，如用表达载体，例如重组噬菌体、质粒或粘粒DNA表达载体转化的细菌；用酵母表达载体转化的酵母；用病毒表达载体(例如，杆状病毒)感染的昆虫细胞系统；用病毒表达载体(例如，花椰菜花叶病毒，CaMV；烟草花叶病毒，TMV)或用细菌表达载体(例如，Ti或pBR322质粒)转化的植物细胞系统；或动物细胞系统，包含哺乳动物细胞，以及更具体地用病毒、质粒、附加型、整合或其它表达载体转化的人细胞系统。因此，某些实施例包含包括编码本文所述多肽，例如融合多肽的多核苷酸序列的表达载体。还包含包括多核苷酸和/或表达载体的宿主细胞。Various expression vector/host systems are known and can be used to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms such as bacteria transformed with expression vectors, eg, recombinant phage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cells infected with viral expression vectors (eg, baculovirus) systems; plant cell systems transformed with viral expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (eg, Ti or pBR322 plasmids); or animal cell systems, including mammalian cells, and more particularly human cell systems transformed with viral, plasmid, episomal, integrative or other expression vectors. Accordingly, certain embodiments include expression vectors comprising polynucleotide sequences encoding polypeptides described herein, eg, fusion polypeptides. Also included are host cells comprising polynucleotides and/or expression vectors.

某些实施例可以采用大肠杆菌类表达系统(参见，例如，结构基因组学联盟等，《自然方法(Nature Methods)》5:135-146,2008)。这些和相关实施例可以部分或完全依赖独立于连接的克隆(LIC)以产生合适的表达载体。在具体实施例中，蛋白质表达可以由T7 RNA聚合酶(例如，pET载体系列)或具有替代启动子(包含例如TAC启动子)的经过修饰的pET载体控制。这些和相关实施例可以利用表达宿主菌株BL21(DE3)，一种支持T7介导的表达并且缺乏用于改善靶蛋白稳定性的lon和ompT蛋白酶的BL21的λDE3溶原菌。还包含携带大肠杆菌中很少使用的编码tRNA的质粒的表达宿主菌株，如ROSETTA^TM(DE3)和Rosetta 2(DE3)菌株。在一些实施例中，可以利用其它大肠杆菌菌株，包含其它大肠杆菌K-12菌株，如W3110(F^-λ^-IN(rrnD-rrnE)1rph-1)和UT5600(F、araC14、leuB6(Am)、secA206(aziR)、lacY1、proC14、tsx67、Δ(ompTfepC)266、entA403、glnX44(AS)、λ^-、trpE38、rfbC1、rpsL109(strR)、xylA5、mtl-1、thiE1),这些大肠杆菌菌株可能导致发酵过程中翻译后修饰水平降低。还可以使用以商标核酸酶和蛋白质提取试剂销售的试剂来改善细胞裂解和样品处理。对于细胞培养，自诱导培养基可以提高许多表达系统的效率，包含高通量表达系统。这种类型的培养基(例如，OVERNIGHT EXPRESS^TM自诱导系统)通过代谢转移逐渐引发蛋白质表达，无需添加如IPTG等人工诱导剂。Certain embodiments may employ an E. coli-like expression system (see, eg, Consortium for Structural Genomics et al., Nature Methods 5:135-146, 2008). These and related examples may rely in part or in full on ligation-independent cloning (LIC) to generate suitable expression vectors. In particular embodiments, protein expression can be controlled by T7 RNA polymerase (eg, the pET vector series) or modified pET vectors with alternative promoters (including, eg, the TAC promoter). These and related examples may utilize the expression host strain BL21(DE3), a λDE3 lysogen that supports T7-mediated expression and lacks BL21 of the lon and ompT proteases for improved target protein stability. Also included are expression host strains carrying tRNA-encoding plasmids rarely used in E. coli, such as the ROSETTA^™ (DE3) and Rosetta 2 (DE3) strains. In some embodiments, other E. coli strains can be utilized, including other E. coli K-12 strains, such as W3110 (F^- λ^- IN(rrnD-rrnE)1rph-1) and UT5600 (F, araC14, leuB6(Am) , secA206(aziR), lacY1, proC14, tsx67, Δ(ompTfepC)266, entA403, glnX44(AS), λ-, trpE38,^rfbC1 , rpsL109(strR), xylA5, mtl-1, thiE1), these E. coli strains May result in reduced levels of post-translational modifications during fermentation. can also be used as a trademark Nucleases and Protein Extraction Reagents sell reagents to improve cell lysis and sample processing. For cell culture, autoinduction media can increase the efficiency of many expression systems, including high-throughput expression systems. This type of medium (eg, the OVERNIGHT EXPRESS^™ Auto-Induction System) gradually induces protein expression by metabolic transfer without the addition of artificial inducers such as IPTG.

特定实施例采用六聚组氨酸标签(如以商标融合物销售的标签)，然后进行固定化金属亲和色谱(IMAC)纯化或相关技术。然而，在某些方面，临床级蛋白质可以从大肠杆菌包涵体中分离，无需或不使用亲和标签(参见，例如，Shimp等人，《蛋白质表达与纯化(Protein Expr Purif.)》50:58-67,2006)。作为另外一个实例，某些实施例可以采用冷激诱导的大肠杆菌高产量生产系统，因为在低温下大肠杆菌中蛋白质的过表达改善了大肠杆菌的溶解度和稳定性(参见，例如，Qing等人，《自然生物技术(Nature Biotechnology)》22:877-882,2004)。Certain embodiments employ a hexahistidine tag (such as the trademark tags for fusion sales) followed by immobilized metal affinity chromatography (IMAC) purification or related techniques. However, in certain aspects, clinical grade proteins can be isolated from E. coli inclusion bodies without or without the use of affinity tags (see, eg, Shimp et al., Protein Expr Purif. 50:58 -67, 2006). As another example, certain embodiments may employ a cold-shock inducible E. coli high-yield production system because overexpression of proteins in E. coli at low temperatures improves E. coli solubility and stability (see, eg, Qing et al. , "Nature Biotechnology" 22:877-882, 2004).

还包含高密度细菌发酵系统。例如，真氧产碱杆菌的高细胞密度培养允许在细胞密度超过150g/L时产生蛋白质，以及在滴度超过10g/L时表达重组蛋白。在酵母酿酒酵母中，可以使用许多含有如α因子、醇氧化酶和PGH等组成型或诱导型启动子的载体。对于评论，参见Ausubel等人(同上)和Grant等人，《酶学方法(Methods Enzymol.)》153:516-544,1987。还包含巴斯德毕赤酵母(Pichia pandoris)表达系统(参见，例如，Li等人，《自然生物技术(Nature Biotechnology.)》24,210-215,2006；以及Hamilton等人，《科学(Science)》，301:1244,2003)。某些实施例包含被工程化以选择性地糖基化蛋白质的酵母系统，包含具有人源化N-糖基化途径的酵母等(参见，例如，Hamilton等人，《科学(Science.)》313:1441-1443,2006；Wildt等人，《微生物自然评论(Nature Reviews Microbiol.)》3:119-28,2005；以及Gerngross等人，《自然生物技术(Nature Biotechnology.)》22:1409-1414,2004；美国专利第7,629,163号；第7,326,681号；和第7,029,872号)。仅举例来说，重组酵母培养物可以在冯巴赫瓶(Fernbach Flask)或15L、50L、100L和200L发酵罐等中生长。Also includes a high-density bacterial fermentation system. For example, high cell density culture of R. eutropha allows protein production at cell densities in excess of 150 g/L, and expression of recombinant proteins at titers in excess of 10 g/L. In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH can be used. For a review, see Ausubel et al. (supra) and Grant et al., Methods Enzymol. 153:516-544, 1987. Also included are the Pichia pandoris expression system (see, eg, Li et al., Nature Biotechnology. 24, 210-215, 2006; and Hamilton et al., Science , 301:1244, 2003). Certain embodiments include yeast systems engineered to selectively glycosylate proteins, including yeast with humanized N-glycosylation pathways, etc. (see, eg, Hamilton et al., Science. 313:1441-1443, 2006; Wildt et al., Nature Reviews Microbiol. 3:119-28, 2005; and Gerngross et al., Nature Biotechnology. 22:1409- 1414,2004; US Patent Nos. 7,629,163; 7,326,681; and 7,029,872). By way of example only, recombinant yeast cultures can be grown in Fernbach Flasks or 15L, 50L, 100L and 200L fermentors and the like.

在使用植物表达载体的情况下，编码多肽的序列的表达可以由许多启动子中的任何一个驱动。例如，如CaMV的35S和19S启动子等病毒启动子可以单独使用或与烟草花叶病毒的ω前导序列组合使用(Takamatsu，《欧洲分子生物学学会杂志(EMBO J.)》6:307-311,1987)。替代性地，可以使用如RUBISCO的小亚基或热休克启动子等的植物启动子(Coruzzi等人，《欧洲分子生物学学会杂志(EMBO J.)》3:1671-1680,1984；Broglie等人,《科学(Science.)》224:838-843,1984；以及Winter等人，《细胞分化的结果与问题(ResultsProbl.Cell Differ.)》17:85-105,1991)。可以通过直接的DNA转化或病原体介导的转染将这些构建体引入植物细胞中。在许多通常可获得的评论(参见，例如，McGraw Hill的Hobbs，《科技年鉴(Yearbook of Science and Technology)》，第191-196页,1992)中描述了此类技术。Where plant expression vectors are used, expression of the sequence encoding the polypeptide can be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader of tobacco mosaic virus (Takamatsu, EMBO J. 6:307-311 , 1987). Alternatively, plant promoters such as the small subunit of RUBISCO or the heat shock promoter can be used (Coruzzi et al., EMBO J. 3:1671-1680, 1984; Broglie et al. Human, Science. 224:838-843, 1984; and Winter et al., Results Probl. Cell Differ. 17:85-105, 1991). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of commonly available reviews (see, eg, McGraw Hill, Hobbs, Yearbook of Science and Technology, pp. 191-196, 1992).

昆虫系统还可以用于表达所关注的多肽。例如，在一个此类系统中，使用苜蓿银纹夜蛾核多角体病毒(AcNPV)作为载体在草地贪夜蛾细胞或粉纹夜蛾细胞中表达外源基因。可以将编码多肽的序列克隆到病毒的非必需区域，如多角体蛋白基因，并置于多角体蛋白启动子的控制下。成功插入多肽编码序列将使多角体蛋白基因失活并产生缺少外壳蛋白的重组病毒。然后可以使用重组病毒来感染例如可以表达所关注的多肽的草地贪夜蛾细胞或粉纹夜蛾细胞(Engelhard等人，《美国科学院院刊(PNAS USA.)》91:3224-3227,1994)。还包含杆状病毒表达系统，包含利用SF9、SF21和T.ni细胞的杆状病毒表达系统(参见，例如，Murphy和Piwnica-Worms，《蛋白质科学现行方案(Curr Protoc Protein Sci.)》，第5章:第5.4单元,2001)。昆虫系统可以提供类似于哺乳动物系统的翻译后修饰。Insect systems can also be used to express polypeptides of interest. For example, in one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or Trichoplusia cells. Sequences encoding polypeptides can be cloned into non-essential regions of the virus, such as the polyhedrin gene, and placed under the control of the polyhedrin promoter. Successful insertion of the polypeptide coding sequence will inactivate the polyhedrin gene and produce recombinant virus lacking the coat protein. The recombinant virus can then be used to infect, for example, Spodoptera frugiperda cells or Trichoplusias cells that can express the polypeptide of interest (Engelhard et al., PNAS USA. 91:3224-3227, 1994) . Also included are baculovirus expression systems, including those utilizing SF9, SF21, and T.ni cells (see, eg, Murphy and Piwnica-Worms, Curr Protoc Protein Sci., p. Chapter 5: Unit 5.4, 2001). Insect systems can provide post-translational modifications similar to mammalian systems.

在哺乳动物宿主细胞中，许多表达系统在本领域熟知并且可以商购获得。示例性哺乳动物载体系统包含例如来自英杰公司(Invitrogen)的pCEP4、pREP4和pREP7，来自库塞尔公司(Crucell)的PerC6系统，和如来自英杰公司的pLP1等基于慢病毒的系统等。例如，在使用腺病毒作为表达载体的情况下，可以将编码所关注的多肽的序列连接到由晚期启动子和三联前导序列组成的腺病毒转录/翻译复合物中。在病毒基因组的非必需E1或E3区域中的插入可以用于获得能够在感染的宿主细胞中表达多肽的活病毒(Logan和Shenk，《美国科学院院刊(PNAS USA.)》81:3655-3659,1984)。另外，如劳氏肉瘤病毒(RSV)增强子等转录增强子可用于在哺乳动物宿主细胞中增加表达。In mammalian host cells, many expression systems are well known in the art and are commercially available. Exemplary mammalian vector systems include, eg, pCEP4, pREP4, and pREP7 from Invitrogen, the PerC6 system from Crucell, and lentivirus-based systems such as pLP1 from Invitrogen, among others. For example, where an adenovirus is used as an expression vector, the sequence encoding the polypeptide of interest can be ligated into an adenovirus transcription/translation complex consisting of a late promoter and a tripartite leader sequence. Insertions in non-essential El or E3 regions of the viral genome can be used to obtain live viruses capable of expressing polypeptides in infected host cells (Logan and Shenk, PNAS USA. 81:3655-3659 , 1984). Additionally, transcriptional enhancers such as the Rous Sarcoma Virus (RSV) enhancer can be used to increase expression in mammalian host cells.

有用的哺乳动物宿主细胞系的实例包含由SV40(COS-7，ATCC CRL 1651)转化的猴肾CV1系；人胚胎肾系(被亚克隆以在悬浮培养物中生长的293或293细胞，Graham等人，《普通病毒学期刊(J.Gen.Virol.)》36:59,1977)；幼仓鼠肾细胞(BHK，ATCC CCL10)；小鼠支持细胞(TM4，Mather，《生殖生物学(Biol.Reprod.)》23:243-251,1980)；猴肾细胞(CV1 ATCCCCL 70)；非洲绿猴肾细胞(VERO-76，ATCC CRL-1587)；人宫颈癌细胞(HELA，ATCC CCL 2)；犬肾细胞(MDCK，ATCC CCL 34)；布法罗大鼠肝细胞(BRL 3A，ATCC CRL 1442)；人肺细胞(W138，ATCC CCL 75)；人肝细胞(Hep G2，HB 8065)；小鼠乳腺肿瘤(MMT 060562，ATCCCCL51)；TR1细胞(Mather等人，《纽约科学院年鉴(Annals N.Y.Acad.Sci.)》383:44-68,1982)；MRC 5细胞；FS4细胞；以及人肝癌细胞系(Hep G2)。其它有用的哺乳动物宿主细胞系包含中国仓鼠卵巢(CHO)细胞，其包含DHFR-CHO细胞(Urlaub等人，《美国科学院院刊(PNASUSA.)》77:4216,1980)；和如NSO和Sp2/0等骨髓瘤细胞系。对于适用于蛋白质生产的某些哺乳动物宿主细胞系的评论，参见，例如，Yazaki和Wu，《分子生物学方法(Methods inMolecular Biology)》，第248卷(B.K.C Lo编辑，Humana出版社，新泽西州托托瓦市，2003)，第255-268页。某些优选的哺乳动物细胞表达系统包含基于CHO和HEK293细胞的表达系统。哺乳动物表达系统可以利用附着的细胞系，例如，在T-烧瓶、滚瓶或细胞工厂，或悬浮培养物中，例如，在本领域已知的1L和5L旋转瓶，5L、14L、40L、100L和200L搅拌槽生物反应器中，或20/50L和100/200L WAVE生物反应器等中。Examples of useful mammalian host cell lines include the monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); the human embryonic kidney line (293 or 293 cells subcloned to grow in suspension culture, Graham et al, J. Gen. Virol. 36:59, 1977); Baby Hamster Kidney Cells (BHK, ATCC CCL10); Mouse Sertoli cells (TM4, Mather, Biol Reprod.)" 23:243-251, 1980); monkey kidney cells (CV1 ATCCCCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2) ; Canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); Human lung cells (W138, ATCC CCL 75); Human hepatocytes (Hep G2, HB 8065); Mouse mammary tumor (MMT 060562, ATCCCCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68, 1982); MRC 5 cells; FS4 cells; and human liver cancer Cell line (Hep G2). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., PNAS USA. 77:4216, 1980); and, for example, NSO and Sp2 /0 and other myeloma cell lines. For a review of certain mammalian host cell lines suitable for use in protein production, see, eg, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (Edited by B.K.C Lo, Humana Press, NJ City of Totova, 2003), pp. 255-268. Certain preferred mammalian cell expression systems include CHO and HEK293 cell-based expression systems. Mammalian expression systems can utilize attached cell lines, e.g., in T-flasks, roller bottles, or cell factories, or in suspension cultures, e.g., 1L and 5L spinner flasks, 5L, 14L, 40L, In 100L and 200L stirred tank bioreactors, or in 20/50L and 100/200L WAVE bioreactors, etc.

另外，宿主细胞株可以由于其调节已插入序列的表达或以期望的方式加工所表达的蛋白质而被选择。多肽的这些修饰包含但不限于翻译后修饰，如乙酰化、羧化、糖基化、磷酸化、脂化和酰化，或插入非天然存在的氨基酸(一般参见美国专利第7,939,496号；第7,816,320号；第7,947,473号；第7,883,866号；第7,838,265号；第7,829,310号；第7,820,766号；第7,820,766号；第7,7737,226号，第7,736,872号；第7,638,299号；第7,632,924号；和第7,230,068号)。切割蛋白质的“前体原”形式的翻译后加工还可以用于促进正确的插入、折叠和/或功能。除细菌细胞以外，还可以选择如酵母、CHO、HeLa、MDCK、HEK293和W138等具有或甚至缺乏此类翻译后活性的特定细胞机械和特征机制的不同宿主细胞，以确保正确修饰和加工外来蛋白质。Additionally, host cell lines can be selected for their modulation of expression of inserted sequences or processing of expressed proteins in a desired manner. These modifications of polypeptides include, but are not limited to, post-translational modifications, such as acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation, or insertion of non-naturally occurring amino acids (see generally U.S. Patent Nos. 7,939,496; 7,816,320 7,947,473; 7,883,866; 7,838,265; 7,829,310; 7,820,766; 7,820,766; ). Post-translational processing of the "pro-pro" form of the cleavage protein can also be used to facilitate correct insertion, folding and/or function. In addition to bacterial cells, different host cells such as yeast, CHO, HeLa, MDCK, HEK293 and W138 can be selected that possess or even lack specific cellular machinery and characteristic mechanisms for such post-translational activity to ensure correct modification and processing of foreign proteins .

用于缀合的示例性方法可以使用标准化学、生物化学和/或分子技术进行第一多肽(例如，ADI、六聚体多肽)与第二多肽(例如，TNF超家族配体、三聚体多肽)或更多多肽的缀合或偶联。如何使用可获得的本领域公认的方法，并根据本公开内容制备缀合物将是显而易见的。在一些情况下，当偶联缀合物的主要组分时，通常优选的是所采用的技术和所得的连接化学物质基本上不会干扰缀合物的单独组分的期望功能或活性。Exemplary methods for conjugation can use standard chemical, biochemical, and/or molecular techniques to perform a first polypeptide (eg, ADI, a hexameric polypeptide) with a second polypeptide (eg, a TNF superfamily ligand, a trimeric polypeptide). Conjugation or coupling of polymeric polypeptides) or more polypeptides. It will be apparent how to prepare conjugates in light of the present disclosure using available art-recognized methods. In some cases, when coupling the major components of the conjugate, it is generally preferred that the technique employed and the resulting linking chemistry do not substantially interfere with the desired function or activity of the individual components of the conjugate.

在某些实施例中，缀合物是融合多肽或融合蛋白。如本文所述和本领域已知的，在一些情况下，融合多肽在表达系统中表达为重组多肽。融合多肽可以含有多肽序列的一个或多个拷贝，并且可以含有以任何期望的排列存在的基于多肽的所关注试剂的一个或多个拷贝。In certain embodiments, the conjugate is a fusion polypeptide or fusion protein. In some cases, fusion polypeptides are expressed as recombinant polypeptides in an expression system, as described herein and known in the art. A fusion polypeptide may contain one or more copies of the polypeptide sequence, and may contain one or more copies of the polypeptide-based agent of interest in any desired arrangement.

对于融合蛋白，编码融合多肽组分和任选的肽连接子组分的DNA序列可以单独组装，然后连接到合适的表达载体中。将编码一种多肽组分的DNA序列的3'端用或不用肽连接子连接到编码一种或多种其它多肽组分的DNA序列的5'端，使得序列的阅读框同相。连接的DNA序列可操作地连接到合适的转录或翻译调节元件。负责DNA表达的调节元件仅位于编码第一多肽的DNA序列的5'端。类似地，结束翻译所需的终止密码子和转录终止信号仅存在于编码最C端多肽的DNA序列的3'端。这允许翻译成保留两种组分多肽的生物活性的单一融合多肽。For fusion proteins, the DNA sequences encoding the fusion polypeptide components and optional peptide linker components can be assembled separately and then ligated into a suitable expression vector. The 3' end of a DNA sequence encoding one polypeptide component is ligated to the 5' end of a DNA sequence encoding one or more other polypeptide components, with or without a peptide linker, such that the reading frames of the sequences are in phase. The linked DNA sequences are operably linked to suitable transcriptional or translational regulatory elements. The regulatory elements responsible for DNA expression are located only 5' to the DNA sequence encoding the first polypeptide. Similarly, stop codons and transcription termination signals required to end translation are only present at the 3' end of the DNA sequence encoding the most C-terminal polypeptide. This allows translation into a single fusion polypeptide that retains the biological activity of the two component polypeptides.

类似的技术，主要是如启动子、终止密码子和转录终止信号等调节元件的布置可以应用于非融合多肽的重组产生，如用于产生非融合缀合物(例如，化学偶联的缀合物)的多肽。Similar techniques, primarily the placement of regulatory elements such as promoters, stop codons, and transcription termination signals, can be applied to the recombinant production of non-fusion polypeptides, such as for the production of non-fusion conjugates (e.g., chemically coupled conjugates). substance) polypeptides.

本公开的多核苷酸和融合多核苷酸可以含有编码多肽序列的核酸的一个或多个拷贝，和/或可以含有编码多肽药剂的核酸的一个或多个拷贝。The polynucleotides and fusion polynucleotides of the present disclosure may contain one or more copies of a nucleic acid encoding a polypeptide sequence, and/or may contain one or more copies of a nucleic acid encoding a polypeptide agent.

在一些实施例中，将编码多肽和/或融合多肽的多核苷酸直接引入宿主细胞中，并在足以诱导一个或多个已编码的多肽的表达的条件下孵育细胞。可以使用本领域技术人员熟知的标准技术并与本文提供的多肽和核酸序列组合来制备本公开的多肽序列。In some embodiments, a polynucleotide encoding a polypeptide and/or fusion polypeptide is introduced directly into a host cell, and the cell is incubated under conditions sufficient to induce expression of one or more encoded polypeptides. The polypeptide sequences of the present disclosure can be prepared using standard techniques well known to those of skill in the art in combination with the polypeptide and nucleic acid sequences provided herein.

因此，根据某些实施例，提供了包括编码本文所述的多肽或融合多肽的多核苷酸或融合多核苷酸的重组宿主细胞。多肽或融合多肽在宿主细胞中的表达可以通过在适当条件下培养含有多核苷酸的重组宿主细胞来实现。在通过表达产生一种或多种多肽后，可以使用任何合适的技术分离和/或纯化所述一种或多种多肽，然后根据需要使用。在本文别处描述了示例性多核苷酸、表达载体和宿主细胞。Thus, according to certain embodiments, there is provided a recombinant host cell comprising a polynucleotide or fusion polynucleotide encoding a polypeptide or fusion polypeptide described herein. Expression of a polypeptide or fusion polypeptide in a host cell can be accomplished by culturing a recombinant host cell containing the polynucleotide under appropriate conditions. Following production of one or more polypeptides by expression, the one or more polypeptides may be isolated and/or purified using any suitable technique and then used as desired. Exemplary polynucleotides, expression vectors and host cells are described elsewhere herein.

可以根据本领域已知的多种技术纯化和表征由重组细胞产生的多肽，例如，融合多肽。用于进行蛋白质纯化和分析蛋白质纯度的示例性系统包含快速蛋白质液相色谱(FPLC)(例如，AKTA和Bio-Rad FPLC系统)、高效液相色谱(HPLC)(例如，Beckman和WatersHPLC)。用于纯化的示例性化学反应包含本领域已知的离子交换色谱(例如，Q、S)、尺寸排阻色谱、盐梯度、亲和纯化(例如，Ni、Co、FLAG、麦芽糖、谷胱甘肽、蛋白质A/G)、凝胶过滤、反相、陶瓷离子交换色谱和疏水相互作用柱(HIC)等。Polypeptides produced by recombinant cells, eg, fusion polypeptides, can be purified and characterized according to a variety of techniques known in the art. Exemplary systems for performing protein purification and analyzing protein purity include fast protein liquid chromatography (FPLC) (eg, AKTA and Bio-Rad FPLC systems), high performance liquid chromatography (HPLC) (eg, Beckman and Waters HPLC). Exemplary chemical reactions for purification include ion exchange chromatography (eg, Q, S), size exclusion chromatography, salt gradients, affinity purification (eg, Ni, Co, FLAG, maltose, glutathione) known in the art Peptide, Protein A/G), Gel Filtration, Reversed Phase, Ceramic Ion exchange chromatography and hydrophobic interaction column (HIC) etc.

在一些实施例中，缀合物是非融合多肽，例如，通过将第一多肽(例如，ADI、六聚体多肽)化学连接或偶联到第二多肽(例如，TNF超家族配体、三聚体多肽)或更多多肽而产生的缀合物。所采用的特定偶联化学将取决于多肽的结构、生物活性剂内多个官能团的潜在存在、保护/去保护步骤的需求、药剂的化学稳定性等，并容易由本领域技术人员确定。用于制备本公开的缀合物的说明性偶联化学可见于例如Wong(1991)，“《蛋白质缀合和交联的化学(Chemistry of Protein Conjugation and Crosslinking)》”，CRC出版社，佛罗里达州波卡拉顿市；以及Brinkley，“《用染料、半抗原和交联试剂制备蛋白质偶联物的方法的简要概述(A Brief Survey of Methods for Preparing Protein Conjugates with Dyes,Haptens,and Crosslinking Reagents)》”，《生物缀合化学(Bioconjug.Chem.)》，3:2013,1992。优选地，由于采用了缀合技术，例如相对于未缀合多肽，缀合物的结合能力和/或活性基本上未降低。In some embodiments, the conjugate is a non-fusion polypeptide, eg, by chemically linking or coupling a first polypeptide (eg, ADI, hexameric polypeptide) to a second polypeptide (eg, a TNF superfamily ligand, trimeric polypeptide) or more polypeptides. The particular coupling chemistry employed will depend on the structure of the polypeptide, the potential presence of multiple functional groups within the bioactive agent, the need for protection/deprotection steps, the chemical stability of the agent, etc., and is readily determined by one skilled in the art. Illustrative coupling chemistry for preparing the conjugates of the present disclosure can be found, for example, in Wong (1991), "Chemistry of Protein Conjugation and Crosslinking", CRC Press, FL Boca Raton; and Brinkley, "A Brief Survey of Methods for Preparing Protein Conjugates with Dyes, Haptens, and Crosslinking Reagents" , "Bioconjug. Chem.", 3:2013, 1992. Preferably, the binding capacity and/or activity of the conjugate is not substantially reduced as a result of the conjugation technique employed, eg, relative to the unconjugated polypeptide.

在某些实施例中，第一多肽(例如，ADI、六聚体多肽)直接或间接地偶联到第二多肽(例如，TNF超家族配体、三聚体多肽)。当每个所关注多肽具有能够与另一个多肽反应的取代基时，两个所关注多肽之间可能发生直接反应。例如，一个多肽上的亲核基团，如氨基或巯基可以能够与另一个多肽上的含羰基的基团(如酸酐或酰卤)或含有良好离去基团的烷基(例如，卤化物)反应。In certain embodiments, a first polypeptide (eg, ADI, hexameric polypeptide) is coupled directly or indirectly to a second polypeptide (eg, TNF superfamily ligand, trimeric polypeptide). A direct reaction between two polypeptides of interest may occur when each polypeptide of interest has a substituent capable of reacting with the other polypeptide. For example, a nucleophilic group such as an amino or sulfhydryl group on one polypeptide may be capable of interacting with a carbonyl-containing group (such as an anhydride or halide) or an alkyl group with a good leaving group (eg, a halide) on another polypeptide. )reaction.

替代性地，如本文所述，可能需要通过如本文所述的连接子基团，包含如本文所述的非肽连接子和肽连接子，间接偶联第一多肽(例如，ADI、六聚体多肽)和第二多肽(例如，TNF超家族配体、三聚体多肽)。连接子基团还可以充当间隔子以使第一多肽和第二多肽间隔开，以避免干扰键合能力、靶向能力或其它功能。连接子基团还可以用于提高多肽上取代基的化学反应性，从而提高偶联效率。化学反应性的提高还可以促进药剂或药剂上的官能团的使用，否则这是不可能的。连接基团的实例包含例如二硫化物基团、硫醚基团、酸不稳定基团、光不稳定基团、肽酶不稳定基团和酯酶不稳定基团。在其它说明性实施例中，缀合物包含连接基团，如美国专利第5,208,020号或欧洲专利0 425235B1以及Chari等人，《癌症研究(Cancer Research.)》52:127-131,1992中公开的连接基团。本文描述了另外的示例性连接子。Alternatively, as described herein, indirect coupling of the first polypeptide (eg, ADI, Hexalinker) may be desired through a linker group as described herein, including non-peptide linkers and peptide linkers as described herein. polymeric polypeptides) and second polypeptides (eg, TNF superfamily ligands, trimeric polypeptides). The linker group can also act as a spacer to separate the first and second polypeptides to avoid interfering with binding ability, targeting ability, or other functions. Linker groups can also be used to increase the chemical reactivity of substituents on polypeptides, thereby increasing coupling efficiency. Increased chemical reactivity can also facilitate the use of agents or functional groups on agents that would not otherwise be possible. Examples of linking groups include, for example, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, and esterase labile groups. In other illustrative embodiments, the conjugate comprises a linking group, as disclosed in US Patent No. 5,208,020 or European Patent No. 0 425235 B1 and Chari et al., Cancer Research. 52:127-131, 1992 the linking group. Additional exemplary linkers are described herein.

在某些示例性实施例中，包括琥珀酰亚胺酯官能团的多肽与包括氨基的多肽之间反应形成酰胺键；包括氧羰基咪唑基官能团的多肽与包括氨基的多肽之间反应形成氨基甲酸酯键；包括对硝基苯基碳酸酯官能团的多肽与包括氨基的多肽之间反应形成氨基甲酸酯键；包括三氯苯基碳酸酯官能团的多肽与包括氨基的多肽之间反应形成氨基甲酸酯键；包括硫代酯官能团的多肽与包括氨基的多肽之间反应形成氨基甲酸酯键；包括丙醛官能团的多肽与包括氨基的多肽之间反应形成仲胺键。In certain exemplary embodiments, the reaction between a polypeptide comprising a succinimidyl ester functional group and a polypeptide comprising an amino group forms an amide bond; the reaction between a polypeptide comprising an oxycarbonyl imidazolyl functional group and a polypeptide comprising an amino group forms a carbamate Ester bond; reaction between a polypeptide including a p-nitrophenyl carbonate functional group and a polypeptide including an amino group to form a carbamate bond; reaction between a polypeptide including a trichlorophenyl carbonate function and a polypeptide including an amino group to form a carbamate Ester bond; reaction between a polypeptide including a thioester functional group and a polypeptide including an amino group to form a carbamate bond; reaction between a polypeptide including a propionaldehyde functional group and a polypeptide including an amino group to form a secondary amine bond.

在一些示例性实施例中，包括丁醛官能团的多肽与包括氨基的多肽之间反应形成仲胺键；包括缩醛官能团的多肽与包括氨基的多肽之间反应形成仲胺键；包括哌啶酮官能团的多肽与包括氨基的多肽之间反应形成仲胺键；包括甲基酮官能团的多肽与包括氨基的多肽之间反应形成仲胺键；包括三氟乙基磺酸酯官能团的多肽与包括氨基的多肽之间反应形成仲胺键；包括马来酰亚胺官能团的多肽与包括氨基的多肽之间反应形成仲胺键；包括醛官能团的多肽与包括氨基的多肽之间反应形成仲胺键；并且包括肼官能团的多肽与包括羧酸基的多肽之间反应形成仲胺键。In some exemplary embodiments, the reaction between a polypeptide including a butyraldehyde functional group and a polypeptide including an amino group forms a secondary amine bond; the reaction between a polypeptide including an acetal functional group and a polypeptide including an amino group forms a secondary amine bond; including piperidones The reaction between the polypeptide including the functional group and the polypeptide including the amino group forms a secondary amine bond; the reaction between the polypeptide including the methyl ketone functional group and the polypeptide including the amino group forms the secondary amine bond; the polypeptide including the trifluoroethanesulfonate functional group and the amino group include A secondary amine bond is formed by the reaction between the polypeptides comprising the maleimide functional group and a secondary amine bond is formed by the reaction between a polypeptide comprising a maleimide functional group and a polypeptide comprising an amino group; a secondary amine bond is formed by the reaction between a polypeptide comprising an aldehyde functional group and a polypeptide comprising an amino group; And the reaction between the polypeptide including the hydrazine functional group and the polypeptide including the carboxylic acid group forms a secondary amine bond.

在特定示例性实施例中，包括马来酰亚胺官能团的多肽与包括硫醇基的多肽之间反应形成硫醚键；包括乙烯基砜官能团的多肽与包括硫醇基的多肽之间反应形成硫醚键；包括硫醇官能团的多肽与包括硫醇基的多肽之间反应形成二硫键；包括正吡啶基二硫化物官能团的多肽与包括硫醇基的多肽之间反应形成二硫键；并且包括碘乙酰胺官能团的多肽与包括硫醇基的多肽之间反应形成硫醚键。In certain exemplary embodiments, the reaction between a polypeptide comprising a maleimide functional group and a polypeptide comprising a thiol group forms a thioether bond; the reaction between a polypeptide comprising a vinyl sulfone functional group and a polypeptide comprising a thiol group forms a thioether bond Thioether bond; a disulfide bond is formed between a polypeptide including a thiol functional group and a polypeptide including a thiol group; a disulfide bond is formed between a polypeptide including an n-pyridyl disulfide functional group and a polypeptide including a thiol group; And a thioether bond is formed between the polypeptide including the iodoacetamide functional group and the polypeptide including the thiol group.

在具体实施例中，胺-巯基交联剂用于制备缀合物。在一个优选实施例中，例如，交联剂是琥珀酰亚胺基-4-(N-马来酰亚胺甲基)环己烷-1-羧酸酯(SMCC)(赛默科技(ThermoScientific))，其是在中等长度的环己烷稳定的间隔臂(8.3埃)的相对端处含有NHS-酯和马来酰亚胺反应性基团的巯基交联剂。SMCC是不可裂解且膜可渗透的交联剂，所述交联剂可以用于创造巯基反应性马来酰亚胺活化剂(例如，多肽)，用于随后与缀合物的组分进行反应。NHS酯在pH 7-9下与伯胺反应形成稳定的酰胺键。马来酰亚胺与pH 6.5-7.5下的巯基反应形成稳定的硫醚键。因此，SMCC的胺反应性NHS酯与多肽的伯胺快速交联，然后所得的巯基反应性马来酰亚胺基可与另一个多肽的半胱氨酸残基反应，以产生特定所关注缀合物。In specific embodiments, amine-thiol crosslinkers are used to prepare conjugates. In a preferred embodiment, for example, the crosslinking agent is succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (Thermo Scientific )), which are sulfhydryl crosslinkers containing NHS-ester and maleimide reactive groups at opposite ends of a medium-length cyclohexane-stabilized spacer arm (8.3 Angstroms). SMCCs are non-cleavable and membrane-permeable cross-linkers that can be used to create sulfhydryl-reactive maleimide activators (eg, polypeptides) for subsequent reaction with the components of the conjugate . NHS esters react with primary amines at pH 7-9 to form stable amide bonds. Maleimide reacts with sulfhydryl groups at pH 6.5-7.5 to form stable thioether linkages. Thus, the amine-reactive NHS ester of SMCC rapidly cross-links with the primary amine of the polypeptide, and the resulting thiol-reactive maleimide group can then react with the cysteine residue of another polypeptide to generate the specific conjugation of interest compound.

在某些具体实施例中，多肽被修饰以含有暴露的巯基以促进交联，例如以促进与马来酰亚胺活化的多肽的交联。在一些具体实施例中，用修饰伯胺的试剂修饰多肽以添加受保护的硫醇巯基。在一些实施例中，试剂N-琥珀酰亚胺基-S-乙酰基硫代乙酸酯(SATA)(赛默科技)用于产生硫醇化多肽。In certain embodiments, the polypeptides are modified to contain exposed sulfhydryl groups to facilitate cross-linking, eg, to facilitate cross-linking with maleimide-activated polypeptides. In some embodiments, the polypeptide is modified with a primary amine-modifying agent to add a protected thiol sulfhydryl group. In some embodiments, the reagent N-succinimidyl-S-acetylthioacetate (SATA) (Thermo Scientific) is used to generate thiolated polypeptides.

在某些实施例中，马来酰亚胺活化的多肽在合适的条件下与硫醇化多肽反应以产生缀合物。应当理解，通过在这些反应中操纵SMCC、SATA、药剂和多肽的比例，可能产生具有不同化学计量、分子量和性质的缀合物。In certain embodiments, maleimide-activated polypeptides are reacted with thiolated polypeptides under suitable conditions to produce conjugates. It will be appreciated that by manipulating the ratio of SMCC, SATA, agent and polypeptide in these reactions, it is possible to generate conjugates with different stoichiometry, molecular weight and properties.

在一些说明性实施例中，使用如N-琥珀酰亚胺基-3-(2-吡啶基二硫代)丙酸酯(SPDP)、琥珀酰亚胺基-4-(N-马来酰亚胺甲基)环己烷-1-羧酸酯、亚氨基四氢噻吩(IT)、亚氨酸酯的双功能衍生物(如己二亚氨盐二甲酯HCL)、活性酯(如辛二酸二琥珀酰亚胺)、醛(如戊二醛)、双-叠氮基化合物(如双(对-叠氮基苯甲酰基)己二胺)、双-重氮基衍生物(如双-(对-重氮基苯甲酰基)-乙二胺)、二异氰酸酯(如2,6-二异氰酸甲苯酯)和双活性氟化合物(如1,5-二氟-2,4-二硝基苯)等双功能蛋白偶联剂制备缀合物。特定的偶联剂包含N-琥珀酰亚胺基-3-(2-吡啶基二硫代)丙酸酯(SPDP)(Carlsson等人，《生物化学期刊(Biochem.J.)》173:723-737[1978])和N-琥珀酰亚胺基-4-(2-吡啶硫基)戊酸酯(SPP),以提供二硫键。In some illustrative embodiments, use such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleyl Iminomethyl)cyclohexane-1-carboxylate, iminotetrahydrothiophene (IT), bifunctional derivatives of imidoesters (such as adipimide dimethyl ester HCL), active esters (such as Suberic acid disuccinimide), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexamethylenediamine), bis-diazo derivatives ( Such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as 2,6-diisocyanate tolyl) and dual reactive fluorine compounds (such as 1,5-difluoro-2, 4-dinitrobenzene) and other bifunctional protein coupling agents to prepare conjugates. Particular coupling agents include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J. 173:723 -737 [1978]) and N-succinimidyl-4-(2-pyridylthio)valerate (SPP) to provide a disulfide bond.

本文讨论的具体交联策略仅是可用于产生本文所述的缀合物的合适缀合策略的许多实例中的一些实例。本领域技术人员将显而易见的是，可以采用多种其它双功能或多功能试剂，包括同功能和异功能试剂(如在伊利诺伊州罗克福德市皮尔斯化学公司(PierceChemical Co.)的目录中描述的试剂)作为连接子基团。偶联可能例如通过氨基、羧基、巯基或氧化的碳水化合物残基而受到影响。有许多参考文献描述了此类方法，例如，Rodwell等人的美国专利第No.4,671,958号。The specific cross-linking strategies discussed herein are only a few of the many examples of suitable conjugation strategies that can be used to generate the conjugates described herein. It will be apparent to those skilled in the art that a variety of other bifunctional or multifunctional reagents can be employed, including homofunctional and heterofunctional reagents (such as those described in the catalogue of Pierce Chemical Co., Rockford, IL). ) as a linker group. Coupling may be effected, for example, through amino groups, carboxyl groups, sulfhydryl groups or oxidized carbohydrate residues. There are numerous references describing such methods, eg, US Patent No. 4,671,958 to Rodwell et al.

缀合物还可以通过各种“点击化学”技术制备，包含模块化、范围广、产率非常高的反应，主要产生可以通过非色谱方法除去并且可以是立体特异性的但不一定是对映选择性的无害副产物(参见Kolb等人，《德国应用化学会刊(Angew Chem Int Ed Engl.)》40:2004-2021,2001)。特定实例包含采用叠氮化物和炔烃的Huisgen 1,3-偶极环加成的缀合技术，还称为“叠氮化物-炔烃环加成”反应(参见Hein等人，《药物研究(Pharm Res.)》25:2216-2230,2008)。叠氮化物-炔烃环加成反应的非限制性实例包含铜催化的叠氮化物-炔烃环加成(CuAAC)反应和钌催化的叠氮化物-炔烃环加成(RuAAC)反应。Conjugates can also be prepared by a variety of "click chemistry" techniques, encompassing modular, broad-ranging, very high-yielding reactions that yield predominantly non-chromatographic methods that can be removed by non-chromatographic methods and can be stereospecific but not necessarily enantiomeric Selective innocuous by-products (see Kolb et al., Angew Chem Int Ed Engl. 40:2004-2021, 2001). Specific examples include conjugation techniques employing Huisgen 1,3-dipolar cycloaddition of azides and alkynes, also known as "azide-alkyne cycloaddition" reactions (see Hein et al., "Drug Research"). (Pharm Res. 25:2216-2230, 2008). Non-limiting examples of azide-alkyne cycloaddition reactions include copper-catalyzed azide-alkyne cycloaddition (CuAAC) reactions and ruthenium-catalyzed azide-alkyne cycloaddition (RuAAC) reactions.

CuAAC在较广的温度范围内工作，对水性条件不敏感，pH范围为4到12，并且耐受宽范围的官能团(参见Himo等人，《美国化学会志(J Am Chem Soc.)》127:210-216,2005)。活性Cu(I)催化剂可以例如使用抗坏血酸钠作为还原剂从Cu(I)盐或Cu(II)盐中产生。这种反应形成1,4-取代的产物，使其具有区域特异性(参见Hein等人，同上)。CuAAC operates over a wide temperature range, is insensitive to aqueous conditions, has a pH range of 4 to 12, and is tolerant of a wide range of functional groups (see Himo et al., J Am Chem Soc. 127 : 210-216, 2005). Active Cu(I) catalysts can be produced from Cu(I) salts or Cu(II) salts, for example, using sodium ascorbate as reducing agent. This reaction forms 1,4-substituted products, making them regiospecific (see Hein et al., supra).

RuAAC利用能够催化叠氮化物到末端炔烃的环加成的五甲基环戊二烯基氯化钌[Cp*RuCl]络合物，区域选择性地产生1,5-二取代的1,2,3-三唑(参见Rasmussen等人，《有机化学通讯(Org.Lett.)》9:5337-5339,2007)。另外，与CuAAC相反，RuAAC还可以与内部炔烃一起使用以提供完全取代的1,2,3-三唑。RuAAC utilizes a pentamethylcyclopentadienylruthenium chloride [Cp*RuCl] complex capable of catalyzing the cycloaddition of azides to terminal alkynes to regioselectively generate 1,5-disubstituted 1,5-disubstituted 1,5-disubstituted 1,5-disubstituted 2,3-triazoles (see Rasmussen et al., Org. Lett. 9:5337-5339, 2007). Additionally, in contrast to CuAAC, RuAAC can also be used with internal alkynes to provide fully substituted 1,2,3-triazoles.

如本文所述，任何一种或多种融合或非融合技术可以用于制备缀合物。As described herein, any one or more fusion or non-fusion techniques can be used to prepare the conjugates.

使用方法和组合物Method of use and composition

还包含使用本文所述的缀合物治疗有需要的受试者的方法，以及包括缀合物的组合物。例如，某些实施例包含在有需要的受试者中治疗癌症、改善癌症症状或抑制癌症进展的方法，包括向受试者施用本文所述的缀合物或包括所述缀合物的组合物。Also included are methods of treating a subject in need thereof using the conjugates described herein, as well as compositions comprising the conjugates. For example, certain embodiments encompass methods of treating cancer, ameliorating the symptoms of cancer, or inhibiting the progression of cancer in a subject in need thereof, comprising administering to the subject a conjugate described herein or a combination comprising the conjugate thing.

本文所述的方法和组合物可以用于治疗任何种类的癌症。在一些实施例中，癌症选自以下中的一个或多个：肝细胞癌(HCC)、黑色素瘤、转移性黑色素瘤、胰腺癌、前列腺癌、小细胞肺癌、间皮瘤、淋巴细胞性白血病、慢性髓细胞性白血病、淋巴瘤、肝癌、肉瘤、白血病、急性髓性白血病、复发性急性髓性白血病、B细胞恶性肿瘤、乳腺癌、卵巢癌、结直肠癌、胃癌、胶质瘤(例如，星形细胞瘤、少突神经胶质瘤、室管膜瘤或脉络丛乳头状瘤)、多形性胶质母细胞瘤(例如，巨细胞胶质母细胞瘤或胶质肉瘤)、脑膜瘤、垂体腺瘤、前庭神经鞘瘤、原发性CNS淋巴瘤、原始神经外胚层肿瘤(成神经管细胞瘤)、非小细胞肺癌(NSCLC)、肾癌、膀胱癌、子宫癌、食道癌、脑癌、头颈癌、宫颈癌、睾丸癌和胃癌。The methods and compositions described herein can be used to treat any kind of cancer. In some embodiments, the cancer is selected from one or more of the following: hepatocellular carcinoma (HCC), melanoma, metastatic melanoma, pancreatic cancer, prostate cancer, small cell lung cancer, mesothelioma, lymphocytic leukemia , chronic myeloid leukemia, lymphoma, liver cancer, sarcoma, leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia, B cell malignancies, breast cancer, ovarian cancer, colorectal cancer, gastric cancer, glioma (e.g. , astrocytoma, oligodendroglioma, ependymoma, or choroid plexus papilloma), glioblastoma multiforme (eg, giant cell glioblastoma or gliosarcoma), meninges Tumor, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), non-small cell lung cancer (NSCLC), kidney cancer, bladder cancer, uterine cancer, esophageal cancer , brain, head and neck, cervical, testicular and gastric cancers.

在一些实施例中，癌症表现出精氨基琥珀酸合成酶-1(ASS-1)的表达和/或活性的降低，或者是精氨基琥珀酸合成酶-1缺陷的表达和/或活性的降低。在这些和相关实施例的一些实施例中，癌症是ADI敏感的或基本上是ADI敏感的。在一些情况下，ASS-1表达或活性的降低是表达和/或活性相对于合适的对照样品(例如，正常细胞或组织)中的表达和/或活性降低约5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％或更多。在某些实施例中，ASS或ASL表达或活性相对于对照样品中的表达或活性降低至少两倍。In some embodiments, the cancer exhibits decreased expression and/or activity of argininosuccinate synthase-1 (ASS-1), or is deficient in argininosuccinate synthase-1 expression and/or activity . In some of these and related embodiments, the cancer is ADI-sensitive or substantially ADI-sensitive. In some cases, the reduction in ASS-1 expression or activity is about a 5%, 10%, 15% reduction in expression and/or activity relative to expression and/or activity in a suitable control sample (eg, normal cells or tissue) , 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more. In certain embodiments, ASS or ASL expression or activity is reduced at least two-fold relative to expression or activity in a control sample.

在一些实施例中，癌症表现出精氨基琥珀酸合成酶-1(ASS-1)的正常或提高的表达和/或活性。在这些和相关实施例的某些实施例中，癌症是ADI抗性的或基本上ADI抗性的，或ADI不敏感的。In some embodiments, the cancer exhibits normal or increased expression and/or activity of argininosuccinate synthase-1 (ASS-1). In certain of these and related embodiments, the cancer is ADI-resistant or substantially ADI-resistant, or ADI-insensitive.

ASS-1表达或活性可以根据本领域的常规技术测量，包含例如，定量PCR、免疫组织化学、蛋白免疫印迹、酶活性测定(例如，用于测量瓜氨酸转化为精氨基琥珀酸盐或精氨基琥珀酸盐转化为精氨酸和富马酸盐的ADI活性测定法)等。ASS-1 expression or activity can be measured according to routine techniques in the art, including, e.g., quantitative PCR, immunohistochemistry, western blotting, enzymatic activity assays (e.g., for measuring the conversion of citrulline to argininosuccinate or arginine). ADI activity assay for the conversion of aminosuccinate to arginine and fumarate), etc.

在一些实施例中，本文所述的方法或组合物将患者的中位生存期增加4周、5周、6周、7周、8周、9周、10周、15周、20周、25周、30周、40周或更长时间。在某些实施例中，本文所述的方法或组合物将患者的中位生存期增加1年、2年、3年或更长时间。在一些实施例中，本文所述的方法或组合物将无进展生存期增加2周、3周、4周、5周、6周、7周、8周、9周、10周或更长时间。在某些实施例中，本文所述的方法或组合物将无进展生存期增加1年、2年、3年或更长时间。In some embodiments, the methods or compositions described herein increase the median survival of a patient by 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks Weeks, 30 weeks, 40 weeks or more. In certain embodiments, the methods or compositions described herein increase a patient's median survival by 1 year, 2 years, 3 years, or more. In some embodiments, the methods or compositions described herein increase progression-free survival by 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or more . In certain embodiments, the methods or compositions described herein increase progression-free survival by 1 year, 2 years, 3 years, or more.

在某些实施例中，施用的组合物足以使肿瘤消退，如肿瘤存活量的统计学显著降低所示，例如，肿瘤质量降低至少10％、20％、30％、40％、50％或更多，或如改变的(例如，具有统计显著性的降低)扫描尺寸所示。在某些实施例中，施用的组合物足以导致稳定的疾病。在某些实施例中，施用的组合物足以导致熟练的临床医生已知的特定疾病适应症的症状的稳定或临床相关性的减轻。In certain embodiments, the administered composition is sufficient to cause tumor regression, as indicated by a statistically significant reduction in tumor survival, eg, at least a 10%, 20%, 30%, 40%, 50% or more reduction in tumor mass more, or as indicated by an altered (eg, statistically significant reduction) scan size. In certain embodiments, the administered composition is sufficient to cause stable disease. In certain embodiments, the administered composition is sufficient to cause stabilization or clinically relevant reduction of symptoms known to the skilled clinician for a particular disease indication.

用于治疗癌症的方法或组合物可以与其它治疗方式结合。例如，本文所述的组合物可以在其它治疗性干预之前、期间或之后施用于受试者，包含对症护理、化学疗法、放射疗法、手术、移植、激素疗法、光动力疗法、抗生素疗法或其任何组合。对症护理包含施用皮质类固醇，以减轻脑水肿、头痛、认知功能障碍和呕吐，以及施用抗惊厥药，以减少癫痫发作。放射治疗包含全脑照射、分次放射治疗和如立体定向放射外科治疗等放射外科治疗，其可以进一步与传统手术相结合。The methods or compositions for treating cancer can be combined with other treatment modalities. For example, the compositions described herein can be administered to a subject before, during, or after other therapeutic interventions, including symptomatic care, chemotherapy, radiation therapy, surgery, transplantation, hormone therapy, photodynamic therapy, antibiotic therapy, or the like any combination. Symptomatic care includes administration of corticosteroids to reduce cerebral edema, headache, cognitive impairment and vomiting, and administration of anticonvulsants to reduce seizures. Radiation therapy includes whole brain irradiation, fractionated radiation therapy, and radiosurgery such as stereotactic radiosurgery, which can be further combined with conventional surgery.

用于鉴定患有本文所述的一种或多种疾病或病症的受试者的方法是本领域已知的。Methods for identifying subjects with one or more of the diseases or disorders described herein are known in the art.

对于体内用途，例如，对于治疗人类疾病或测试，本文所述的缀合物通常在施用前掺入到一种或多种药物或治疗组合物中。在一些实例中，药物或治疗组合物包括与生理学上可接受的载剂或赋形剂组合的一种或多种本文所述的缀合物。For in vivo use, eg, for treatment of human disease or testing, the conjugates described herein are typically incorporated into one or more pharmaceutical or therapeutic compositions prior to administration. In some examples, a pharmaceutical or therapeutic composition includes one or more conjugates described herein in combination with a physiologically acceptable carrier or excipient.

为了制备药物或治疗组合物，将有效或期望量的一种或多种缀合物与本领域技术人员已知的任何一种或多种药物载剂或赋形剂混合，以适合于特定缀合物和/或施用方式。药物载剂可以是液体、半液体或固体。用于肠胃外、皮内、皮下或局部施用的溶液或悬浮液可以包含，例如，无菌稀释剂(如水)、盐水溶液(例如，磷酸盐缓冲盐水；PBS)、固定油、聚乙二醇、甘油、丙二醇或其它合成溶剂；抗菌剂(如苯甲醇和对羟基苯甲酸甲酯)；抗氧化剂(如抗坏血酸和亚硫酸氢钠)和螯合剂(如乙二胺四乙酸(EDTA))；缓冲剂(如乙酸盐、柠檬酸盐和磷酸盐)。如果静脉内施用(例如，通过IV输注)，则合适的载剂包含生理盐水或磷酸盐缓冲盐水(PBS)，以及含有如葡萄糖、聚乙二醇、聚丙二醇和其混合物的增稠剂和增溶剂的溶液。To prepare a pharmaceutical or therapeutic composition, an effective or desired amount of one or more conjugates is admixed with any one or more pharmaceutical carriers or excipients known to those skilled in the art to suit a particular conjugate composition and/or mode of administration. Pharmaceutical carriers can be liquid, semi-liquid or solid. Solutions or suspensions for parenteral, intradermal, subcutaneous or topical administration may contain, for example, sterile diluents (eg, water), saline solutions (eg, phosphate-buffered saline; PBS), fixed oils, polyethylene glycols , glycerol, propylene glycol or other synthetic solvents; antimicrobials (such as benzyl alcohol and methylparaben); antioxidants (such as ascorbic acid and sodium bisulfite) and chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); Buffers (eg acetate, citrate and phosphate). If administered intravenously (eg, by IV infusion), suitable carriers include physiological saline or phosphate buffered saline (PBS), and thickeners such as dextrose, polyethylene glycol, polypropylene glycol, and mixtures thereof, and Solubilizer solution.

在某些方面，组合物的pH接近生理pH或约pH 7.4，包含约pH 6.5、约7.0、约7.1、约7.2、约7.3、约7.4、约7.5、约7.6、约7.7、约7.8、约7.9、约8.0、约8.5或其任何范围。在具体实施例中，组合物具有一种或多种以下纯度测定：通过SEC HPLC测定，小于约1EU内毒素/mg蛋白、小于约100ng宿主细胞蛋白/mg蛋白、小于约10pg宿主细胞DNA/mg蛋白和/或大于约95％单峰纯度。In certain aspects, the pH of the composition is close to physiological pH or about pH 7.4, including about pH 6.5, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.5, or any range thereof. In specific embodiments, the composition has one or more of the following purity assays: less than about 1 EU endotoxin/mg protein, less than about 100 ng host cell protein/mg protein, less than about 10 pg host cell DNA/mg as determined by SEC HPLC Protein and/or greater than about 95% unimodal purity.

可通过各种不同途径实现施用，包含口服、肠胃外、鼻内、静脉内、皮内、肌肉内、鞘内、皮下、舌下、口腔、直肠、阴道和局部。优选的施用方式取决于待治疗或预防的病症的性质。特定实施例包含通过IV输注施用。Administration can be achieved by a variety of different routes, including oral, parenteral, intranasal, intravenous, intradermal, intramuscular, intrathecal, subcutaneous, sublingual, buccal, rectal, vaginal, and topical. The preferred mode of administration depends on the nature of the condition to be treated or prevented. Particular embodiments include administration by IV infusion.

载剂可以包含，例如，在所采用的剂量和浓度下，对暴露于其中的细胞或哺乳动物无毒的药物上可接受的载剂、赋形剂或稳定剂。生理学上可接受的载剂通常是含水pH缓冲溶液。生理学上可接受的载剂的实例包含如磷酸盐、柠檬酸盐、和其它有机酸等缓冲液；包含抗坏血酸的抗氧化剂；低分子量(少于约10个残基)的多肽；如血清白蛋白、明胶或免疫球蛋白等蛋白质；如聚乙烯吡咯烷酮等亲水聚合物；如甘氨酸、谷氨酰胺、天冬酰胺、精氨酸或赖氨酸等氨基酸；包含葡萄糖、甘露糖、或糊精的单糖、二糖和其它碳水化合物；如EDTA等螯合剂；如甘露醇或山梨醇等糖醇；如钠等成盐抗衡离子；和/或如聚山梨醇酯20(TWEEN^TM)聚乙二醇(PEG)和泊咯沙姆(PLURONICS^TM)等非离子表面活性剂。The carrier may comprise, for example, a pharmaceutically acceptable carrier, excipient or stabilizer that is nontoxic to the cells or mammals to which it is exposed, at the dosages and concentrations employed. Physiologically acceptable carriers are usually aqueous pH buffered solutions. Examples of physiologically acceptable carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; such as serum albumin , proteins such as gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; containing glucose, mannose, or dextrin Monosaccharides, disaccharides, and other carbohydrates; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or polyethylene glycol such as polysorbate 20 (TWEEN^™ ) Nonionic surfactants such as alcohol (PEG) and poloxamer (PLURONICS^™ ).

在一些实施例中，在胶体药物递送系统中(例如，脂质体、白蛋白微球、微乳、纳米颗粒和纳米胶囊)或在粗乳液中，可以例如通过凝聚技术或通过界面聚合(例如，分别为羟甲基纤维素或明胶-微胶囊和聚-(甲基-甲基丙烯酸酯)微胶囊)将一种或多种缀合物包埋在制备的微胶囊中。此类技术在《雷明顿药物科学(Remington's PharmaceuticalSciences)》，第16版，Oslo A.编辑，(1980)中公开。一种或多种颗粒或脂质体可以进一步包括其它治疗剂或诊断剂。In some embodiments, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions, it is possible, for example, by coacervation techniques or by interfacial polymerization (e.g., , hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl-methacrylate) microcapsules, respectively) encapsulate one or more conjugates in the prepared microcapsules. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th ed., ed. by Oslo A., (1980). The one or more particles or liposomes may further include other therapeutic or diagnostic agents.

因此，施用这些和相关药物组合物的典型途径包含但不限于口服、局部、透皮、吸入、肠胃外、舌下、口腔、直肠、阴道和鼻内。本文所使用的术语肠胃外包含皮下注射、静脉内、肌肉内、胸骨内注射或输注技术。某些药物或治疗组合物被配制成允许在将组合物施用给患者时其中含有的有效成分是生物可利用的。将施用于受试者或患者的组合物可以采取一个或多个剂量单位的形式，其中例如，片剂可以是单剂量单位，并且本文所述的气雾剂形式的缀合物的容器可以容纳多个剂量单位。制备这种剂型的实际方法对于本领域技术人员来说是已知的、或者是显而易见的；例如，参见《雷明顿：药学技术与实践(The Science andPractice of Pharmacy》，第20版(费城医药科学学院，2000)。待施用的组合物通常含有治疗有效量的本文所述的缀合物，用于治疗所关注疾病或病症。Thus, typical routes of administration of these and related pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injection, intravenous, intramuscular, intrasternal injection or infusion techniques. Certain pharmaceutical or therapeutic compositions are formulated to allow the active ingredient contained therein to be bioavailable when the composition is administered to a patient. The composition to be administered to a subject or patient can take the form of one or more dosage units, wherein, for example, a tablet can be a single dosage unit and a container of a conjugate described herein in an aerosol form can hold Multiple dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia Pharmacy). Academy of Sciences, 2000). Compositions to be administered typically contain a therapeutically effective amount of a conjugate described herein for the treatment of a disease or condition of interest.

药物或治疗组合物可以是固体或液体形式。在一个实施例中，一种或多种载剂是颗粒状的，使得组合物是例如片剂或粉末形式。一种或多种载剂可以是液体，而组合物是例如，口服油、可注射液体或气雾剂，其可用于例如，吸入施用。当用于口服施用时，药物组合物优选为固体或液体形式，其中半-固体、半-液体、悬浮液和凝胶形式包含在本文所述的为固体或液体的形式中。The pharmaceutical or therapeutic composition can be in solid or liquid form. In one embodiment, the one or more carriers are granular, such that the composition is in tablet or powder form, for example. One or more carriers can be liquids and the compositions are, for example, oral oils, injectable liquids or aerosols, which can be used, for example, for administration by inhalation. When used for oral administration, the pharmaceutical compositions are preferably in solid or liquid form, wherein semi-solid, semi-liquid, suspension and gel forms are included in the solid or liquid forms described herein.

作为用于口服施用的固体组合物，可以将药物组合物配制成粉末、颗粒、压缩片剂、丸剂、胶囊、口香糖、薄片等。这种固体组合物通常含有一种或多种惰性稀释剂或可食用载剂。此外，可能存在以下一种或多种：如羧甲基纤维素、乙基纤维素、微晶纤维素、黄蓍胶或明胶等粘合剂；如淀粉、乳糖或糊精等赋形剂，如海藻酸、海藻酸钠、羧甲基淀粉钠、玉米淀粉等崩解剂；如硬脂酸镁或氢化蓖麻油等润滑剂；如胶体二氧化硅等助流剂；如蔗糖或糖精等甜味剂；如薄荷、水杨酸甲酯或橙味调味品等调味剂；以及着色剂。当药物组合物为胶囊形式时，例如，明胶胶囊，除了上述类型的物质外，所述药物还可以含有如聚乙二醇或油等液体载剂。As solid compositions for oral administration, the pharmaceutical compositions can be formulated into powders, granules, compressed tablets, pills, capsules, chewing gums, flakes, and the like. Such solid compositions typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, tragacanth or gelatin; excipients such as starch, lactose or dextrin, Disintegrants such as alginic acid, sodium alginate, sodium carboxymethyl starch, corn starch; lubricants such as magnesium stearate or hydrogenated castor oil; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin Flavoring agents; flavoring agents such as mint, methyl salicylate, or orange flavoring; and coloring agents. When the pharmaceutical composition is in the form of a capsule, eg, a gelatin capsule, the pharmaceutical composition may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or an oil.

药物或治疗组合物可以是液体形式，例如，酏剂、糖浆、溶液、乳液或悬浮液。作为两个实例，液体可以用于口服施用或通过注射递送。当用于口服施用时，除本发明化合物外，优选的组合物还含有一种或多种甜味剂、防腐剂、染料/着色剂和增味剂。在旨在通过注射施用的组合物中，可以包含表面活性剂、防腐剂、湿润剂、分散剂、悬浮剂、缓冲剂、稳定剂和等渗剂中的一种或多种。Pharmaceutical or therapeutic compositions can be in liquid form, eg, elixirs, syrups, solutions, emulsions, or suspensions. Liquids can be used for oral administration or delivered by injection, as two examples. When used for oral administration, preferred compositions contain, in addition to a compound of the present invention, one or more sweetening agents, preservatives, dyes/colorants and flavor enhancers. In compositions intended for administration by injection, one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers and isotonic agents may be included.

液体药物或治疗组合物，无论其是溶液、悬浮液或其它类似形式，可以包含一种或多种以下佐剂：如注射用水、盐水溶液、优选生理盐水、林格氏溶液、等渗氯化钠等无菌稀释剂，如可用作溶剂或悬浮培养基的合成甘油单酯或甘油二酯、聚乙二醇、甘油、丙二醇或其它溶剂等固定油；如苯甲醇或对羟基苯甲酸甲酯等抗菌剂；如抗坏血酸或亚硫酸氢钠等抗氧化剂；如乙二胺四乙酸等螯合剂；如乙酸盐、柠檬酸盐或磷酸盐等缓冲剂，以及如氯化钠或葡萄糖等用于调节张力的药剂。肠胃外制剂可以封装在由玻璃或塑料制成的安瓶、一次性注射器或多剂量小瓶中。生理盐水是优选佐剂。可注射药物组合物优选地是无菌的。Liquid pharmaceutical or therapeutic compositions, whether in solution, suspension or other similar form, may contain one or more of the following adjuvants such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic chloride Sterile diluents such as sodium, fixed oils such as synthetic mono- or diglycerides, polyethylene glycol, glycerol, propylene glycol, or other solvents, which can be used as solvents or suspension media; such as benzyl alcohol or methylparaben Antibacterial agents such as esters; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffering agents such as acetate, citrate or phosphate; A drug used to adjust tension. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is the preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

用于肠胃外或口服施用的液体药物或治疗组合物应含有一定量的缀合物，以便获得合适的剂量。通常，这个量为组合物中所关注缀合物的至少0.01％。当用于口服施用时，这个量可以在0.1％到约70％的组合物重量之间变化。某些口服药物组合物含有约4％到约75％的所关注缀合物。在某些实施例中，制备药物组合物和制剂，使得肠胃外剂量单位在稀释前含有0.01％到10％的所关注缀合物重量。Liquid pharmaceutical or therapeutic compositions for parenteral or oral administration should contain the conjugate in an amount to obtain a suitable dosage. Typically, this amount is at least 0.01% of the conjugate of interest in the composition. When used for oral administration, this amount may vary from 0.1% to about 70% by weight of the composition. Certain oral pharmaceutical compositions contain from about 4% to about 75% of the conjugate of interest. In certain embodiments, pharmaceutical compositions and formulations are prepared such that a parenteral dosage unit, prior to dilution, contains from 0.01% to 10% by weight of the conjugate of interest.

药物组合物可以旨在用于局部施用，在这种情况下，载剂可以适当地包括溶液、乳剂、乳膏或凝胶基质。例如，基质可以包括以下一种或多种：矿脂、羊毛脂、聚乙二醇、蜂蜡、矿物油、如水和醇等稀释剂、以及乳化剂和稳定剂。增稠剂可以存在于用于局部施用的药物组合物中。如果用于透皮施用，则所述组合物可以包含透皮贴剂或离子电渗疗法装置。The pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, cream or gel base. For example, the base may include one or more of the following: petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents can be present in pharmaceutical compositions for topical administration. If for transdermal administration, the composition may comprise a transdermal patch or an iontophoresis device.

药物组合物可以用于例如栓剂形式的直肠施用，其将在直肠中融化并释放药物。用于直肠施用的组合物可以含有作为合适的无刺激性赋形剂的油性基质。此类基质包含但不限于羊毛脂、可可脂和聚乙二醇。The pharmaceutical composition can be used for rectal administration, eg, in the form of a suppository, which will melt in the rectum and release the drug. Compositions for rectal administration may contain an oily base as a suitable non-irritating excipient. Such bases include, but are not limited to, lanolin, cocoa butter, and polyethylene glycols.

药物组合物可以包含各种材料，这些材料改变固体或液体剂量单位的物理形式。例如，组合物可以包含在活性成分周围形成包衣壳的材料。形成包衣壳的材料通常是惰性的，并且可以选自例如，糖、虫胶和其它肠溶包衣剂。替代性地，可以将活性成分包裹在明胶胶囊中。固体或液体形式的药物组合物可以包含结合到缀合物的组分，从而有助于缀合物的递送。可以以这种能力起作用的合适组分包含单克隆或多克隆抗体、一种或多种蛋白质或脂质体。Pharmaceutical compositions can contain various materials that alter the physical form of the solid or liquid dosage unit. For example, the compositions may contain materials that form a shell around the active ingredient. The material forming the coating shell is generally inert and can be selected from, for example, sugar, shellac and other enteric coating agents. Alternatively, the active ingredient may be enclosed in gelatin capsules. Pharmaceutical compositions in solid or liquid form may contain components bound to the conjugate to facilitate delivery of the conjugate. Suitable components that can function in this capacity include monoclonal or polyclonal antibodies, one or more proteins, or liposomes.

药物组合物可以基本上由剂量单位组成，其可以作为气雾剂施用。术语气雾剂用于表示从胶体性质的系统到由加压包装组成的系统的各种系统。可以通过液化或压缩气体或通过分配活性成分的合适泵系统进行递送。气雾剂可以以单相、双-相或三-相系统递送，以递送一种或多种活性成分。气雾剂的递送包含必要的容器、活化剂、阀门、子容器等，这些可以一起形成试剂盒。本领域普通技术人员无需过多实验即可确定优选的气雾剂。The pharmaceutical composition may consist essentially of dosage units, which may be administered as an aerosol. The term aerosol is used to denote a variety of systems ranging from systems of colloidal nature to those consisting of pressurized packs. Delivery can be by liquefied or compressed gas or by a suitable pump system that dispenses the active ingredient. Aerosols can be delivered in monophasic, biphasic or triphasic systems to deliver one or more active ingredients. The delivery of the aerosol contains the necessary containers, activators, valves, sub-containers, etc., which together can form a kit. One of ordinary skill in the art can determine the preferred aerosol without undue experimentation.

本文所述的组合物可以用保护缀合物不会从身体中快速消除(如定时释放调配物或包衣)的载剂制备。此类载剂包含控释调配物，如但不限于植入物和微囊化递送系统，和可生物降解的生物相容性聚合物，如乙烯醋酸乙烯酯、聚酸酐、聚乙醇酸、聚原酸酯、聚乳酸以及本领域普通技术人员已知的其它物质。The compositions described herein can be prepared with carriers that will protect the conjugate against rapid elimination from the body (eg, time release formulations or coatings). Such carriers include controlled release formulations such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, poly Orthoesters, polylactic acid, and others known to those of ordinary skill in the art.

药物组合物可以通过制药领域熟知的方法制备。例如，用于通过注射施用的药物组合物可以包括一种或多种盐、缓冲剂和/或稳定剂，并与无菌蒸馏水一起形成溶液。可以加入表面活性剂以促进均相溶液或悬浮液的形成。表面活性剂是与缀合物非共价相互作用的化合物，以促进缀合物在含水递送系统中的溶解或均匀悬浮。Pharmaceutical compositions can be prepared by methods well known in the art of pharmacy. For example, a pharmaceutical composition for administration by injection may include one or more salts, buffers and/or stabilizers and form a solution with sterile distilled water. Surfactants can be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that interact non-covalently with the conjugate to facilitate dissolution or uniform suspension of the conjugate in an aqueous delivery system.

组合物可以以治疗有效量施用，其将根据多种因素而变化，包含所用的特定化合物的活性；化合物的代谢稳定性和作用时间；患者的年龄、体重、总体健康、性别和饮食；施用方式和时间；排泄率；药物组合；特定障碍或病症的严重程度；以及受试者正在接受的治疗。The compositions can be administered in therapeutically effective amounts, which will vary depending on a number of factors, including the activity of the particular compound used; the metabolic stability and duration of action of the compound; the patient's age, weight, general health, sex, and diet; the mode of administration and time; excretion rate; drug combination; the severity of the particular disorder or condition; and the treatment the subject is receiving.

精确的剂量和治疗持续时间是所治疗疾病的函数，并且可以使用已知的测试方案凭经验确定，或者通过在本领域已知的模型系统中测试组合物并从中推断。也可以进行受控的临床试验。剂量也可以随着要缓解的病症的严重程度而变化。通常配制和施用药物组合物以发挥治疗上有用的效果，同时最小化不希望的副作用。所述组合物可以一次施用，或者可以分成许多较小的剂量而每隔一段时间施用。对于任何特定受试者，可根据个体需要随时间调整特定剂量方案。The exact dosage and duration of treatment are a function of the disease being treated and can be determined empirically using known testing protocols, or by testing and inferring the composition in model systems known in the art. Controlled clinical trials may also be conducted. The dosage may also vary with the severity of the condition to be alleviated. Pharmaceutical compositions are typically formulated and administered to exert therapeutically useful effects while minimizing undesired side effects. The composition may be administered at one time, or may be divided into a number of smaller doses administered at intervals. For any particular subject, the particular dosage regimen can be adjusted over time according to individual needs.

在一些实施例中，本文所述的组合物的治疗有效量或治疗剂量是有效降低或稳定肿瘤生长的量。在某些情况下，用小剂量开始治疗，然后可以小幅增加所述小剂量，直到达到这种情况下的最佳效果。在一些情况下，治疗有效日剂量(对于70kg哺乳动物)为约0.001mg/kg(即，约0.07mg)到约100mg/kg(即，约7.0g)；优选地，治疗有效剂量(对于70kg哺乳动物)为约0.01mg/kg(即，约0.7mg)到约50mg/kg(即，约3.5g)；更优选地，治疗有效剂量(对于70kg哺乳动物)为约1mg/kg(即，约70mg)到约25mg/kg(即，约1.75g)。In some embodiments, a therapeutically effective amount or therapeutic dose of a composition described herein is an amount effective to reduce or stabilize tumor growth. In some cases, treatment is started with a small dose, which can then be increased in small increments until the optimal effect in the case is achieved. In some instances, the therapeutically effective daily dose (for a 70 kg mammal) is about 0.001 mg/kg (ie, about 0.07 mg) to about 100 mg/kg (ie, about 7.0 g); preferably, the therapeutically effective dose (for a 70 kg) mammal) is about 0.01 mg/kg (ie, about 0.7 mg) to about 50 mg/kg (ie, about 3.5 g); more preferably, the therapeutically effective dose (for a 70 kg mammal) is about 1 mg/kg (ie, about 70 mg) to about 25 mg/kg (ie, about 1.75 g).

在一些实施例中，剂量从约每天施用一次到约每两周或三周施用一次。例如，在某些实施例中，剂量为约每1、2、3、4、5、6或7天施用一次，或约每周施用一次，或约每周两次，或约每周三次，或约每两周或三周一次。In some embodiments, the dose is administered from about once daily to about once every two or three weeks. For example, in certain embodiments, the dose is administered about once every 1, 2, 3, 4, 5, 6, or 7 days, or about once a week, or about twice a week, or about three times a week, Or about every two or three weeks.

在一些实施例中，剂量为约0.1mg/kg到约20mg/kg，或到约10mg/kg，或到约5mg/kg，或到约3mg/kg。在一些实施例中，剂量为约0.10mg/kg、0.15mg/kg、0.20mg/kg、0.25mg/kg、0.30mg/kg、0.35mg/kg、0.40mg/kg、0.45mg/kg、0.50mg/kg、0.55mg/kg、0.60mg/kg、0.65mg/kg、0.70mg/kg、0.75mg/kg、0.80mg/kg、0.85mg/kg、0.90mg/kg、0.95mg/kg、1.0mg/kg、1.5mg/kg、2.0mg/kg、2.5mg/kg、3.0mg/kg、3.5mg/kg、4.0mg/kg、4.5mg/kg、5.0mg/kg、5.5mg/kg、6.0mg/kg、6.5mg/kg、7.0mg/kg、7.5mg/kg、8.0mg/kg、8.5mg/kg、9.0mg/kg、9.5mg/kg、10mg/kg、11mg/kg、12mg/kg、13mg/kg、14mg/kg、15mg/kg、16mg/kg、17mg/kg、18mg/kg、19mg/kg或20mg/kg，包括其间的所有整数和范围。在具体实施例中，剂量为每周一次静脉注射2ml约1mg/kg到每3天一次约20mg/kg。In some embodiments, the dose is about 0.1 mg/kg to about 20 mg/kg, or to about 10 mg/kg, or to about 5 mg/kg, or to about 3 mg/kg. In some embodiments, the dose is about 0.10 mg/kg, 0.15 mg/kg, 0.20 mg/kg, 0.25 mg/kg, 0.30 mg/kg, 0.35 mg/kg, 0.40 mg/kg, 0.45 mg/kg, 0.50 mg/kg mg/kg, 0.55mg/kg, 0.60mg/kg, 0.65mg/kg, 0.70mg/kg, 0.75mg/kg, 0.80mg/kg, 0.85mg/kg, 0.90mg/kg, 0.95mg/kg, 1.0 mg/kg, 1.5mg/kg, 2.0mg/kg, 2.5mg/kg, 3.0mg/kg, 3.5mg/kg, 4.0mg/kg, 4.5mg/kg, 5.0mg/kg, 5.5mg/kg, 6.0 mg/kg, 6.5mg/kg, 7.0mg/kg, 7.5mg/kg, 8.0mg/kg, 8.5mg/kg, 9.0mg/kg, 9.5mg/kg, 10mg/kg, 11mg/kg, 12mg/kg , 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg or 20 mg/kg, including all integers and ranges therebetween. In a specific embodiment, the dose is about 1 mg/kg 2 ml intravenously once weekly to about 20 mg/kg once every 3 days.

还包含包括一种或多种本文所述的缀合物或组合物的患者护理试剂盒。某些试剂盒还包括一种或多种药学上可接受的稀释剂或溶剂，如水(例如无菌水)。在一些实施例中，缀合物储存在小瓶、盒、双室注射器和/或预填充混合系统中。Also included are patient care kits that include one or more of the conjugates or compositions described herein. Certain kits also include one or more pharmaceutically acceptable diluents or solvents, such as water (eg, sterile water). In some embodiments, the conjugates are stored in vials, cartridges, dual chamber syringes and/or prefilled mixing systems.

本文的试剂盒还可以包含一种或多种附加的治疗剂(例如，缀合物)或其它适合于或期望用于正在治疗的适应症或用于期望的诊断应用的组分。本文的试剂盒还可以包含一个或多个注射器或其它促进预期递送模式所需或期望的组分(例如，支架、可植入的贮库等)。The kits herein may also contain one or more additional therapeutic agents (eg, conjugates) or other components suitable or desired for the indication being treated or for the desired diagnostic application. The kits herein may also contain one or more syringes or other components necessary or desired to facilitate the intended mode of delivery (eg, stents, implantable depots, etc.).

本说明书中引用的所有出版物、专利申请和发布的专利均通过引用并入本文，如同每个单独的出版物、专利申请或发布的专利被明确且单独地指明通过引用并入一样。All publications, patent applications and issued patents cited in this specification are herein incorporated by reference as if each individual publication, patent application or issued patent were expressly and individually indicated to be incorporated by reference.

尽管上述发明已经出于清楚理解的目的通过说明和举例的方式进行了一些详细描述，但是根据本公开的教导，对于本领域普通技术人员而言应当容易了解到，可以在不偏离随附权利要求书的精神或范围的情况下对本发明进行某些改变和修改。以下实例仅通过说明的方式而非限制的方式提供。本领域的技术人员应当容易识别可以被改变或修改以产生基本上类似的结果的各种非关键参数。While the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art from the teachings of this disclosure that one can do so without departing from the appended claims. Certain changes and modifications may be made to the invention without departing from the spirit or scope of the book. The following examples are provided by way of illustration only and not by way of limitation. Those skilled in the art should readily identify various non-critical parameters that can be changed or modified to produce substantially similar results.

实例example

实例1Example 1

ADI-PEG 20和TRAIL的组合Combination of ADI-PEG 20 and TRAIL

通过用ADI-PEG 20(用K112E和P210S取代修饰的、来自人型支原体的聚乙二醇化精氨酸脱亚胺酶)、人rhTRAIL或其组合处理各种癌细胞系来测试ADI增加TRAIL抗癌活性的能力，并且在一些情况下反之亦然。所述细胞测定如实例1中所描述的进行。ADI was tested to increase TRAIL resistance by treating various cancer cell lines with ADI-PEG 20 (pegylated arginine deiminase from Mycoplasma hominis modified with K112E and P210S substitutions), human rhTRAIL, or a combination thereof. cancer activity, and in some cases vice versa. The cellular assays were performed as described in Example 1.

ADI敏感(大多数ASS1表达低或检测不到)癌细胞系的结果如下表E1所示。Results for ADI-sensitive (most ASS1 expression low or undetectable) cancer cell lines are shown in Table E1 below.

这些结果说明在多种癌细胞系的细胞杀伤中，相对于单独的ADI-PEG 20和/或rhTRAIL，ADI-PEG 20与rhTRAIL组合之间的协同或加和作用。这些结果还说明ADI-PEG 20增强了rhTRAIL在以其它方式对rhTRAIL具有抗性的癌细胞系中的活性。在多种癌细胞系中观察到显著或协同增加的细胞杀伤活性，包含乳腺癌细胞、伯基特淋巴瘤细胞、结肠癌细胞、胶质母细胞瘤癌细胞、白血病细胞、黑色素瘤癌细胞、非小细胞肺癌(NSCLC)细胞、卵巢癌细胞、胰腺癌细胞、前列腺癌细胞和肾癌细胞。These results demonstrate a synergistic or additive effect between ADI-PEG 20 and rhTRAIL in combination relative to ADI-PEG 20 and/or rhTRAIL alone in cell killing of various cancer cell lines. These results also demonstrate that ADI-PEG 20 enhances the activity of rhTRAIL in cancer cell lines that are otherwise resistant to rhTRAIL. Significantly or synergistically increased cell killing activity was observed in a variety of cancer cell lines, including breast cancer cells, Burkitt lymphoma cells, colon cancer cells, glioblastoma cancer cells, leukemia cells, melanoma cancer cells, Non-small cell lung cancer (NSCLC) cells, ovarian cancer cells, pancreatic cancer cells, prostate cancer cells and kidney cancer cells.

图1A-1D进一步说明了相对于单独的ADI-PEG 20和/或rhTRAIL，ADI-PEG 20和rhTRAIL组合对各种癌细胞系的相对活力的协同作用。图1A示出了HCT116结肠癌细胞系的协同作用，图1B示出了Caki-1肾癌细胞系的协同作用，图1C示出了ACHN肾癌细胞系的协同作用，并且图1D示出了A375黑色素瘤细胞系的协同作用。Figures 1A-1D further illustrate the synergistic effect of the combination of ADI-PEG 20 and rhTRAIL on the relative viability of various cancer cell lines relative to ADI-PEG 20 and/or rhTRAIL alone. Figure 1A shows the synergy of the HCT116 colon cancer cell line, Figure 1B shows the synergy of the Caki-1 kidney cancer cell line, Figure 1C shows the synergy of the ACHN kidney cancer cell line, and Figure 1D shows the synergy of the Caki-1 kidney cancer cell line Synergy of the A375 melanoma cell line.

图2A-2C表明了与Raji Burkitt淋巴瘤细胞系中单独的每种药剂相比，ADI-PEG20和rhTRAIL对半胱天冬酶3/7活化(图2A)、细胞死亡诱导(图2B)以及未致力于细胞凋亡的活细胞百分比降低(半胱天冬酶3/7未活化的细胞；图2C)的协同作用。在用检测活化的半胱天冬酶3/7和死亡细胞的荧光试剂(来自赛默飞世尔科技(ThermoFisher Scientific)的CellEvent半胱天冬酶3/7试剂盒)染色后，通过流式细胞术分析测定死亡细胞或具有和不具有活化的半胱天冬酶3/7的细胞的百分比。Figures 2A-2C show caspase 3/7 activation (Figure 2A), induction of cell death (Figure 2B) and induction of cell death (Figure 2B) by ADI-PEG20 and rhTRAIL compared to each agent alone in Raji Burkitt's lymphoma cell line. Synergistic effect of reduced percentage of viable cells not committed to apoptosis (caspase 3/7 inactivated cells; Figure 2C). After staining with fluorescent reagents that detect activated caspase 3/7 and dead cells (CellEvent Caspase 3/7 kit from ThermoFisher Scientific), flow cytometry Cytometry analysis determined the percentage of dead cells or cells with and without activated caspase 3/7.

下表E2中示出了抗ADI的(ASS1的相对高表达)癌细胞系的结果。Results for ADI-resistant (relatively high expression of ASS1) cancer cell lines are shown in Table E2 below.

表E2中的结果示出在某些抗ADI细胞系中，ADI-PEG 20不一定增强rhTRAIL的癌细胞杀伤活性(由于高ASS1)(反之亦然)。然而，在一些抗ADI癌细胞系中(例如，MDA-MB-453和H1975，其中ADI具有至少某种最小活性)，ADI-PEG 20和rhTRAIL的组合相对于单独的ADI-PEG 20和/或TRAIL在癌细胞杀伤活性方面表现出协合作用、增强作用和/或相互作用。实例2和3示出了ADI的一些生物活性，这些生物活性可能有助于ADI增强或协同TRAIL的能力。The results in Table E2 show that ADI-PEG 20 did not necessarily enhance the cancer cell killing activity of rhTRAIL (due to high ASS1) in certain anti-ADI cell lines (and vice versa). However, in some anti-ADI cancer cell lines (eg, MDA-MB-453 and H1975, in which ADI has at least some minimal activity), the combination of ADI-PEG 20 and rhTRAIL was more effective than ADI-PEG 20 and/or ADI-PEG 20 alone. TRAIL exhibits synergy, potentiation and/or interaction in cancer cell killing activity. Examples 2 and 3 show some of the biological activities of ADI that may contribute to the ability of ADI to enhance or synergize with TRAIL.

实例2Example 2

ADI-PEG 20上调DR5受体ADI-PEG 20 upregulates the DR5 receptor

为了探索ADI增加或增强rhTRAIL活性的潜在机制，在用ADI-PEG 20处理癌细胞系后，通过流式细胞术测量DR4和DR5受体的表达。To explore the underlying mechanism by which ADI increases or enhances rhTRAIL activity, the expression of DR4 and DR5 receptors was measured by flow cytometry after treatment of cancer cell lines with ADI-PEG 20.

实验工作流程由细胞处理、收集和用可固定的存活/死亡染色剂(赛默飞世尔)染色，以及抗体在冰上识别TRAIL受体30分钟组成。然后用多色流式细胞仪洗去未掺入的存活/死亡染料和未结合的抗体并进行分析。存活/死亡染色剂和抗体用在流式细胞仪的不同通道中检测到的不同荧光团标记。同型对照抗体用于评估和控制非特异性结合。受体表达在单线态和活细胞门控的细胞群中进行分析。The experimental workflow consisted of cell processing, harvesting and staining with fixable live/death stain (Thermo Fisher), and antibody recognition of TRAIL receptors for 30 min on ice. Unincorporated live/dead dye and unbound antibody were then washed away with a multicolor flow cytometer and analyzed. Survival/death stains and antibodies are labeled with different fluorophores detected in different channels of the flow cytometer. Isotype control antibodies were used to assess and control for nonspecific binding. Receptor expression was analyzed in singlet and live cell-gated cell populations.

图3A-3D示出在Panc-1、Jurkat、Raji、K562、O-786、ACHN、Caki-1、Caki-2、WM-115和HCT116细胞系中DR5受体的表达通过ADI-PEG 20上调。在这些细胞系中DR4受体的表达较低或检测不到，并且没有明显受到ADI-PEG 20处理的影响。Figures 3A-3D show that DR5 receptor expression is upregulated by ADI-PEG 20 in Panc-1, Jurkat, Raji, K562, O-786, ACHN, Caki-1, Caki-2, WM-115 and HCT116 cell lines . Expression of the DR4 receptor was low or undetectable in these cell lines and was not significantly affected by ADI-PEG 20 treatment.

实例3Example 3

ADI-PEG 20下调存活蛋白ADI-PEG 20 down-regulates survivin

图4表明了ADI敏感细胞系用ADI-PEG 20处理后存活蛋白水平降低。存活蛋白已被证明能阻止TRAIL的活性。因此，降低存活蛋白水平(连同DR5上调)可能有助于ADI增强和/或增加癌细胞系中TRAIL的凋亡活性的能力。Figure 4 shows that ADI-sensitive cell lines were treated with ADI-PEG 20 to reduce survivin levels. Survivin has been shown to block the activity of TRAIL. Therefore, reducing survivin levels (along with DR5 upregulation) may contribute to the ability of ADI to enhance and/or increase the apoptotic activity of TRAIL in cancer cell lines.

实例4Example 4

ADI和TRAIL的缀合物Conjugates of ADI and TRAIL

来自鸽支原体的精氨酸脱亚胺酶(ADI)与人TNF相关凋亡诱导配体(TRAIL)的胞外结构域(残基114-281)之间的融合蛋白根据常规技术克隆、表达和纯化，然后针对抗癌活性对其进行测试。下表E3提供了测试的ADI-TRAIL融合蛋白的总结。A fusion protein between arginine deiminase (ADI) from Mycoplasma pigeonii and the extracellular domain (residues 114-281) of human TNF-related apoptosis-inducing ligand (TRAIL) was cloned, expressed and Purified and then tested for anticancer activity. Table E3 below provides a summary of the ADI-TRAIL fusion proteins tested.

另外，来自其它六聚体物种和其交换结构域的ADI用于利用GGGGS连接子(SEQ IDNO:76)制备含有人TRAIL(aa 114-281)的融合蛋白。下表E4提供了所产生的六聚体ADI-TRAIL融合蛋白以及作为ADI-TRAIL融合蛋白的一部分的ADI六聚体的活性的总结。Additionally, ADI from other hexameric species and their exchange domains was used to prepare fusion proteins containing human TRAIL (aa 114-281) using the GGGGS linker (SEQ ID NO:76). Table E4 below provides a summary of the activity of the hexameric ADI-TRAIL fusion proteins produced and the ADI hexamers that are part of the ADI-TRAIL fusion protein.

为了评估融合蛋白对癌细胞生长、活力和凋亡诱导的作用，将各种癌细胞系接种在96孔板中并暴露于融合蛋白。在接种后立即处理悬浮细胞系，同时使贴壁细胞附着过夜，然后向培养物中加入研究性蛋白质处理剂。To assess the effect of fusion proteins on cancer cell growth, viability, and induction of apoptosis, various cancer cell lines were seeded in 96-well plates and exposed to fusion proteins. Suspension cell lines were treated immediately after seeding while adherent cells were allowed to attach overnight before the investigational protein treatment was added to the culture.

通过将来自测试样品的细胞活力信号除以未处理对照的细胞活力信号来计算相对细胞活力。用检测活细胞的试剂(如刃天青或CellTiter-Glo(普洛麦格(Promega)))测定、并用酶标仪(比色、荧光或发光信号)测量细胞活力。对于细胞凋亡，使用普洛麦格的半胱天冬酶3/7Glo试剂和酶标仪的发光读数来评估半胱天冬酶3/7活化。半胱天冬酶3/7活化和细胞活力还通过用检测活化的半胱天冬酶3/7和死亡细胞的荧光试剂(来自赛默飞世尔科技的CellEvent半胱天冬酶3/7试剂盒)染色的细胞的流式细胞术分析来评估。Relative cell viability was calculated by dividing the cell viability signal from the test sample by the cell viability signal of the untreated control. Cell viability is measured with a reagent that detects viable cells, such as resazurin or CellTiter-Glo (Promega), and with a microplate reader (colorimetric, fluorescent or luminescent signal). For apoptosis, caspase 3/7 activation was assessed using Promega's Caspase 3/7 Glo reagent and luminescence readout from a microplate reader. Caspase 3/7 activation and cell viability were also detected by fluorescent reagents (CellEvent Caspase 3/7 from Thermo Fisher Scientific, which detect activated caspase 3/7 and dead cells). kit) was assessed by flow cytometry analysis of stained cells.

图5描绘了示例性ADI-TRAIL或TRAIL-ADI融合蛋白示意图。Figure 5 depicts a schematic representation of exemplary ADI-TRAIL or TRAIL-ADI fusion proteins.

图6A-6C示出了相对于单独的rhTRAIL、单独的M.col.ADI和作为单独多肽的rhTRAIL和M.col.ADI的组合，示例性M.col.ADI-TRAIL融合多肽与L1连接子(见表E3)对抗ADI的Colo 205癌细胞系中半胱天冬酶3/7诱导(图6A)和相对细胞活力(图6B和6C)的作用。在该细胞系(高ASS1表达)中，ADI不具有显着活性，并且癌细胞杀伤活性归因于ADI-TRAIL融合多肽的TRAIL组分。Figures 6A-6C show exemplary M.col.ADI-TRAIL fusion polypeptides with the L1 linker relative to rhTRAIL alone, M.col.ADI alone, and a combination of rhTRAIL and M.col.ADI as individual polypeptides (See Table E3) Effects of caspase 3/7 induction (Fig. 6A) and relative cell viability (Figs. 6B and 6C) in ADI-resistant Colo 205 cancer cell lines. In this cell line (high ASS1 expression), ADI had no significant activity, and the cancer cell killing activity was attributed to the TRAIL component of the ADI-TRAIL fusion polypeptide.

图7A-7C示出了相对于单独的rhTRAIL、单独的M.col.ADI以及作为单独多肽的rhTRAIL和M.col.ADI的组合，示例性M.col.ADI-TRAIL融合多肽与L1连接子(见表E3)对ADI敏感的HCT116肿瘤细胞系中半胱天冬酶3/7诱导(图7A)和相对细胞活力(图7B和7C)的作用。Figures 7A-7C show exemplary M.col.ADI-TRAIL fusion polypeptides with an L1 linker relative to rhTRAIL alone, M.col.ADI alone, and a combination of rhTRAIL and M.col.ADI as individual polypeptides (See Table E3) Effects of caspase 3/7 induction (Fig. 7A) and relative cell viability (Figs. 7B and 7C) in the ADI-sensitive HCT116 tumor cell line.

图8A-8B示出了相对于单独的rhTRAIL、单独的M.col.ADI和作为单独多肽的rhTRAIL和M.col.ADI的组合，示例性M.col.ADI-TRAIL融合多肽与L1连接子(见表E3)对ADI敏感的Jurkat肿瘤细胞系中半胱天冬酶3/7诱导(图8A)和相对细胞活力(图8B)的作用。图9A-9D示出了来自表E3的示例性M.col.ADI-TRAIL融合多肽对抗ADI的Colo 205癌细胞系中半胱天冬酶3/7诱导(图9A和9B)和相对细胞活力(图9C和9D)的作用。图10A-10C示出了来自表E3的示例性M.col.ADI-TRAIL融合多肽对ADI敏感的HCT116细胞系中半胱天冬酶3/7诱导(图10A)和相对细胞活力(图10B-10C)的作用。图11A-11B示出了来自表E3的示例性M.col.ADI-TRAIL融合多肽对ADI敏感的Jurkat细胞系中半胱天冬酶3/7诱导(图11A)和相对细胞活力(图11B)的作用。Figures 8A-8B show exemplary M.col.ADI-TRAIL fusion polypeptides with the L1 linker relative to rhTRAIL alone, M.col.ADI alone, and a combination of rhTRAIL and M.col.ADI as individual polypeptides (See Table E3) Effects of caspase 3/7 induction (FIG. 8A) and relative cell viability (FIG. 8B) in ADI-sensitive Jurkat tumor cell lines. Figures 9A-9D show caspase 3/7 induction (Figures 9A and 9B) and relative cell viability in the ADI-resistant Colo 205 cancer cell line of exemplary M.col. ADI-TRAIL fusion polypeptides from Table E3 (FIGS. 9C and 9D). Figures 10A-10C show caspase 3/7 induction (Figure 10A) and relative cell viability (Figure 10B) in an ADI-sensitive HCT116 cell line by exemplary M.col. ADI-TRAIL fusion polypeptides from Table E3 -10C). FIGS. 11A-11B show caspase 3/7 induction ( FIG. 11A ) and relative cell viability ( FIG. 11B ) in an ADI-sensitive Jurkat cell line by exemplary M.col. ADI-TRAIL fusion polypeptides from Table E3 ) effect.

图9-11示出了来自表E1的示例性M.col.ADI-TRAIL融合多肽在ADI敏感和抗ADI的三种不同细胞系中具有相似的活性。Figures 9-11 show that exemplary M.col. ADI-TRAIL fusion polypeptides from Table E1 have similar activity in three different cell lines, ADI sensitive and ADI resistant.

下表E5总结了在抗ADI的Colo 205细胞系和ADI敏感的HCT116细胞系中半胱天冬酶3/7诱导和相对细胞活力降低的情况下来自表E4的示例性ADI-TRAIL融合多肽的IC₅₀值。这些数据表明示例性ADI-TRAIL融合多肽具有相似的活性。Table E5 below summarizes the effects of exemplary ADI-TRAIL fusion polypeptides from Table E4 upon caspase 3/7 induction and reduced relative cell viability in the ADI-resistant Colo 205 cell line and the ADI-sensitive HCT116 cell line._IC50 value. These data demonstrate that exemplary ADI-TRAIL fusion polypeptides have similar activity.

图12A-12C示出了在M.col.ADI(K192C或K287C)中具有一个或多个点突变的示例性M.col.ADI-TRAIL融合多肽(包含非聚乙二醇化的和用2K或20K PEG聚乙二醇化的M.col.ADI-TRAIL融合多肽)对抗ADI的Colo 205细胞系中半胱天冬酶3/7诱导(图12A)和相对细胞活力(图12B-12C)的作用。图13A-13C示出了在M.col.ADI(K192C或K287C)中具有点突变的示例性M.col.ADI-TRAIL融合多肽(包含非聚乙二醇化的和用2K或20K PEG聚乙二醇化的M.col.ADI-TRAIL融合多肽)对ADI敏感的HCT116细胞系中半胱天冬酶3/7诱导(图13A)和相对细胞活力(图13B-13C)的作用。上述聚乙二醇化构建体与非聚乙二醇化构建体中ADI酶活性相似。Figures 12A-12C show exemplary M.col.ADI-TRAIL fusion polypeptides (comprising non-PEGylated and Effects of 20K PEG PEGylated M.col. ADI-TRAIL fusion polypeptide) on caspase 3/7 induction (FIG. 12A) and relative cell viability (FIGS. 12B-12C) in ADI-resistant Colo 205 cell line . Figures 13A-13C show exemplary M.col.ADI-TRAIL fusion polypeptides (comprising non-PEGylated and PEGylated with 2K or 20K PEG) with point mutations in M.col.ADI (K192C or K287C) Effects of diolated M.col. ADI-TRAIL fusion polypeptide) on caspase 3/7 induction (FIG. 13A) and relative cell viability (FIGS. 13B-13C) in the ADI-sensitive HCT116 cell line. The ADI enzymatic activity in the pegylated constructs described above was similar to the non-pegylated constructs.

图14A-14C示出了示例性TRAIL-M.col.ADI与M.col.ADI-TRAIL融合多肽对抗ADI的Colo 205细胞系中半胱天冬酶3/7诱导(图14A)和相对细胞活力(图14B-14C)的作用。因为这种Colo 205细胞系对ADI活性具有抗性，所以观察到的半胱天冬酶3/7活化和随后的活力降低是由于TRAIL部分的促细胞凋亡活性。如图14A-14C所示，TRAIL-M.col.ADI融合蛋白与M.col.ADI-TRAIL融合蛋白相比，TRAIL活性有所提高(约2倍)。Figures 14A-14C show caspase 3/7 induction (Figure 14A) and relative cells in ADI-resistant Colo 205 cell line with exemplary TRAIL-M.col.ADI and M.col.ADI-TRAIL fusion polypeptides The effect of viability (FIGS. 14B-14C). Because this Colo 205 cell line is resistant to ADI activity, the observed caspase 3/7 activation and subsequent reduction in viability is due to the pro-apoptotic activity of the TRAIL moiety. As shown in Figures 14A-14C, the TRAIL-M.col.ADI fusion protein had increased TRAIL activity (about 2-fold) compared to the M.col.ADI-TRAIL fusion protein.

图15A-15C示出了示例性TRAIL-M.col.ADI与M.col.ADI-TRAIL融合多肽对ADI敏感的HCT116细胞系中半胱天冬酶3/7诱导(图15A)和相对细胞活力(图15B-15C)的作用。在这种细胞系中，这两种融合蛋白在诱导半胱天冬酶介导的细胞凋亡方面具有相同的效力。ADI和TRAIL在HCT116细胞系中是协同的。从该实验和其它实验(数据未显示)来看，ADI可以将TRAIL效用增强到一定水平，并且ADI和TRAIL的组合效用不会受到TRAIL部分的效力微小变化的显著影响。换句话说，已经观察到ADI与较低效TRAIL制剂的更强协同作用，并且所述组合效用具有一定的阈值，即使在TRAIL的最优或次优制剂的情况下所述组合效用也会达到所述阈值。Figures 15A-15C show caspase 3/7 induction (Figure 15A) and relative cells in the ADI-sensitive HCT116 cell line by exemplary TRAIL-M.col.ADI and M.col.ADI-TRAIL fusion polypeptides The effect of viability (FIGS. 15B-15C). In this cell line, the two fusion proteins were equally potent in inducing caspase-mediated apoptosis. ADI and TRAIL are synergistic in the HCT116 cell line. From this and other experiments (data not shown), ADI can enhance the efficacy of TRAIL to a certain level, and the combined efficacy of ADI and TRAIL is not significantly affected by small changes in the potency of the TRAIL fraction. In other words, stronger synergy of ADI with less potent TRAIL formulations has been observed, and there is a threshold for the combined utility to be reached even with optimal or suboptimal formulations of TRAIL the threshold.

图16A-16B示出了静脉施用单剂量30mg/kg后，M.col.ADI-TRAIL在CD-1小鼠血清中随时间的PK曲线。在单次注射融合蛋白后，在CD-1小鼠的血清中测量M.col.ADI-TRAIL蛋白水平(图16A-16B)、精氨酸水平和瓜氨酸水平(图16A)以及针对融合蛋白M.col.ADI-TRAIL以及其组分M.col.ADI和rhTRAIL(图16B)的抗体滴度。Figures 16A-16B show the PK profile of M.col. ADI-TRAIL in CD-1 mouse serum over time following intravenous administration of a single dose of 30 mg/kg. M.col. ADI-TRAIL protein levels (FIG. 16A-16B), arginine levels and citrulline levels (FIG. 16A) were measured in the serum of CD-1 mice following a single injection of the fusion protein and for fusion Antibody titers for the protein M.col.ADI-TRAIL and its components M.col.ADI and rhTRAIL (Figure 16B).

血清中融合蛋白的浓度通过ELISA(图16A-16B)进行评估。还评估了血清样品中ADI和TRAIL部分的生物活性，并根据掺入幼稚血清的标准测定了生物活性蛋白的浓度。生物活性蛋白(基于ADI和TRAIL活性)的浓度与通过ELISA法测定的总ADI-TRAIL蛋白非常相似。The concentration of fusion protein in serum was assessed by ELISA (Figures 16A-16B). The biological activity of ADI and TRAIL fractions in serum samples was also assessed, and the concentration of biologically active proteins was determined according to standards spiked into naive serum. The concentrations of biologically active protein (based on ADI and TRAIL activity) were very similar to total ADI-TRAIL protein determined by ELISA.

图17A-17F表明了M.col.ADI-TRAIL在HCT116异种移植模型中的功效。为雌性无胸腺裸小鼠皮下接种HCT116细胞。接种后第7天，将小鼠随机分入处理组(各组之间具有相似的起始肿瘤体积)，并通过静脉注射施用rhTRAIL、M.col.ADI、M.col.ADI-TRAIL融合蛋白或媒剂对照(PBS缓冲液)。每天使用rhTRAIL进行处理，连续5天(肿瘤植入后第7-11天)。在肿瘤植入后第7天和第15天注射M.col.ADI和M.col.ADI-TRAIL融合蛋白。融合蛋白未引起任何明显的体重减轻(图17A)，并且能够减少肿瘤生长(图17B-17F)。*p<0.05，**p<0.01，***p<0.001。通过双向ANOVA评估了与媒剂处理对照组相比融合蛋白处理组中肿瘤减少的统计显著性。Figures 17A-17F demonstrate the efficacy of M.col. ADI-TRAIL in the HCT116 xenograft model. Female athymic nude mice were inoculated subcutaneously with HCT116 cells. On day 7 post-inoculation, mice were randomized into treatment groups (with similar starting tumor volumes between groups) and administered rhTRAIL, M.col.ADI, M.col.ADI-TRAIL fusion protein by intravenous injection or vehicle control (PBS buffer). Treatment with rhTRAIL was performed daily for 5 consecutive days (days 7-11 after tumor implantation). M.col.ADI and M.col.ADI-TRAIL fusion proteins were injected on days 7 and 15 after tumor implantation. The fusion protein did not cause any significant weight loss (Figure 17A) and was able to reduce tumor growth (Figures 17B-17F). *p<0.05, **p<0.01, ***p<0.001. Statistical significance of tumor reduction in the fusion protein-treated group compared to the vehicle-treated control group was assessed by two-way ANOVA.

如图18A-18B所示，血清M.col.ADI-TRAIL与肿瘤体积呈负相关。通过ELISA(总蛋白)和生物测定(活性蛋白)测量的融合蛋白浓度彼此相似。肿瘤植入后第21天和第28天提取血清。检测到的血清总融合蛋白在两个时间点之间相关。图18D中示出了这些血清样品中的精氨酸和瓜氨酸水平。与M.col.ADI-TRAIL组相比，M.col.ADI组血清中瓜氨酸水平更高，精氨酸水平更低。这可能是因为融合蛋白由于其TRAIL部分而定位于肿瘤部位，从而降低了其血清水平。肿瘤体积与血清M.col.ADI-TRAIL之间的反向相关性支持该假设。As shown in Figures 18A-18B, serum M.col. ADI-TRAIL was inversely correlated with tumor volume. The fusion protein concentrations measured by ELISA (total protein) and bioassay (active protein) were similar to each other. Serum was extracted on days 21 and 28 after tumor implantation. The total serum fusion protein detected was correlated between the two time points. Arginine and citrulline levels in these serum samples are shown in Figure 18D. Compared with the M.col.ADI-TRAIL group, the M.col.ADI group had higher serum citrulline levels and lower arginine levels. This may be because the fusion protein localized to the tumor site due to its TRAIL moiety, thereby reducing its serum levels. The inverse correlation between tumor volume and serum M.col.ADI-TRAIL supports this hypothesis.

图19表明了用M.col.ADI-TRAIL处理后HCT116异种移植模型中的剂量依赖性肿瘤生长减少。为雌性无胸腺裸小鼠皮下接种HCT116细胞。接种后第9天，将小鼠随机分入处理组(各组之间具有相似的起始肿瘤体积)，并通过静脉注射施用M.col.ADI-TRAIL融合蛋白或媒剂对照(PBS缓冲液)。M.col.ADI-TRAIL剂量组如下：90mg/kg、30mg/kg、10mg/kg和5mg/kg。前三组仅在肿瘤植入后第9天给药，5mg/kg组在肿瘤植入后第9天和第12天给药。Figure 19 shows a dose-dependent reduction in tumor growth in a HCT116 xenograft model following treatment with M.col. ADI-TRAIL. Female athymic nude mice were inoculated subcutaneously with HCT116 cells. On day 9 post-inoculation, mice were randomized into treatment groups (with similar starting tumor volumes between groups) and administered either the M.col. ADI-TRAIL fusion protein or vehicle control (PBS buffer) by intravenous injection. ). The M.col. ADI-TRAIL dose groups were as follows: 90 mg/kg, 30 mg/kg, 10 mg/kg and 5 mg/kg. The first three groups were administered only on day 9 after tumor implantation, and the 5 mg/kg group was administered on days 9 and 12 after tumor implantation.

双向ANOVA分析揭示了M.col.ADI-TRAIL处理后肿瘤体积减少的统计显著性。处理组与媒剂对照相比P值如下：Two-way ANOVA analysis revealed a statistically significant reduction in tumor volume following M.col. ADI-TRAIL treatment. The P values for the treatment groups compared to the vehicle control are as follows:

第12天：10mg/kg组、30mg/kg组和90mg/kg组，p<0.0001；5mg/kg组，p＝0.0001。Day 12: 10 mg/kg group, 30 mg/kg group and 90 mg/kg group, p<0.0001; 5 mg/kg group, p=0.0001.

第14天：所有组，p<0.0001Day 14: All groups, p<0.0001

第16天：所有组，p<0.0001Day 16: All groups, p<0.0001

在第16天(处理开始后第7天)，高剂量组与低剂量组之间也存在统计学上显著的差异：There was also a statistically significant difference between the high-dose and low-dose groups at day 16 (day 7 after treatment initiation):

·90mg/kg组与10mg/kg组比较：p＝0.047790mg/kg group vs. 10mg/kg group: p=0.0477

·90mg/kg组与5mg/kg组比较：p＝0.001490mg/kg group vs. 5mg/kg group: p=0.0014

·30mg/kg组与5mg/kg组比较：p＝0.0247。• 30 mg/kg group vs. 5 mg/kg group: p=0.0247.

序列表 sequence listing

<110> TDW集团（TDW Group）<110> TDW Group (TDW Group)

Almassy, Robert J. Almassy, Robert J.

Brin, Elena Brin, Elena

Showalter, Richard E. Showalter, Richard E.

Thomson, James A. Thomson, James A.

<120> 蛋白质缀合物<120> Protein Conjugates

<130> TDWG-007/01WO 319744-2053<130> TDWG-007/01WO 319744-2053

<150> US 62/478,398<150> US 62/478,398

<151> 2017-03-29<151> 2017-03-29

<160> 97<160> 97

<170> PatentIn版本3.5<170> PatentIn Version 3.5

<210> 1<210> 1

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<212> PRT<212> PRT

<213> 人型支原体（Mycoplasma hominis）<213> Mycoplasma hominis

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Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser MetGly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser Met

385 390 395 400385 390 395 400

Pro Leu Ser Arg Lys Asp Val Lys TrpPro Leu Ser Arg Lys Asp Val Lys Trp

405 405

<210> 3<210> 3

<211> 392<211> 392

<212> PRT<212> PRT

<213> 海豹脑支原体（Mycoplasma phocicerebrale）<213> Seal Mycoplasma phocicerebrale

<400> 3<400> 3

Ile His Val Tyr Ser Glu Ile Gly Glu Leu Glu Thr Val Leu Val HisIle His Val Tyr Ser Glu Ile Gly Glu Leu Glu Thr Val Leu Val His

1 5 10 151 5 10 15

Glu Pro Gly Arg Glu Ile Asp Tyr Ile Thr Pro Ala Arg Leu Asp GluGlu Pro Gly Arg Glu Ile Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu

20 25 30 20 25 30

Leu Leu Phe Ser Ala Ile Leu Glu Ser His Asp Ala Arg Lys Glu HisLeu Leu Phe Ser Ala Ile Leu Glu Ser His Asp Ala Arg Lys Glu His

35 40 45 35 40 45

Gln Ser Phe Val Lys Gln Leu Lys Asp Asn Gly Ile Asn Val Val GluGln Ser Phe Val Lys Gln Leu Lys Asp Asn Gly Ile Asn Val Val Glu

50 55 60 50 55 60

Leu Thr Asp Leu Val Ala Glu Thr Phe Asp Leu Ala Ser Lys Glu GluLeu Thr Asp Leu Val Ala Glu Thr Phe Asp Leu Ala Ser Lys Glu Glu

65 70 75 8065 70 75 80

Gln Glu Lys Leu Ile Glu Glu Phe Leu Glu Asp Ser Glu Pro Val LeuGln Glu Lys Leu Ile Glu Glu Phe Leu Glu Asp Ser Glu Pro Val Leu

85 90 95 85 90 95

Ser Glu Ala His Lys Thr Ala Val Arg Lys Phe Leu Thr Ser Arg LysSer Glu Ala His Lys Thr Ala Val Arg Lys Phe Leu Thr Ser Arg Lys

100 105 110 100 105 110

Ser Thr Arg Glu Met Val Glu Phe Met Met Ala Gly Ile Thr Lys TyrSer Thr Arg Glu Met Val Glu Phe Met Met Ala Gly Ile Thr Lys Tyr

115 120 125 115 120 125

Asp Leu Gly Ile Glu Ala Asp His Glu Leu Ile Val Asp Pro Met ProAsp Leu Gly Ile Glu Ala Asp His Glu Leu Ile Val Asp Pro Met Pro

130 135 140 130 135 140

Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser Val Gly Asn Gly ValAsn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser Val Gly Asn Gly Val

145 150 155 160145 150 155 160

Thr Ile His Tyr Met Arg Tyr Lys Val Arg Gln Arg Glu Thr Leu PheThr Ile His Tyr Met Arg Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe

165 170 175 165 170 175

Ser Arg Phe Val Phe Ser Asn His Pro Lys Leu Val Lys Thr Pro TrpSer Arg Phe Val Phe Ser Asn His Pro Lys Leu Val Lys Thr Pro Trp

180 185 190 180 185 190

Tyr Tyr Asp Pro Ala Met Lys Met Ser Ile Glu Gly Gly Asp Val PheTyr Tyr Asp Pro Ala Met Lys Met Ser Ile Glu Gly Gly Asp Val Phe

195 200 205 195 200 205

Ile Tyr Asn Asn Asp Thr Leu Val Val Gly Val Ser Glu Arg Thr AspIle Tyr Asn Asn Asp Thr Leu Val Val Gly Val Ser Glu Arg Thr Asp

210 215 220 210 215 220

Leu Glu Thr Ile Thr Leu Leu Ala Lys Asn Ile Lys Ala Asn Lys GluLeu Glu Thr Ile Thr Leu Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu

225 230 235 240225 230 235 240

Val Glu Phe Lys Arg Ile Val Ala Ile Asn Val Pro Lys Trp Thr AsnVal Glu Phe Lys Arg Ile Val Ala Ile Asn Val Pro Lys Trp Thr Asn

245 250 255 245 250 255

Leu Met His Leu Asp Thr Trp Leu Thr Met Leu Asp Lys Asp Lys PheLeu Met His Leu Asp Thr Trp Leu Thr Met Leu Asp Lys Asp Lys Phe

260 265 270 260 265 270

Leu Tyr Ser Pro Ile Ala Asn Asp Val Phe Lys Phe Trp Asp Tyr AspLeu Tyr Ser Pro Ile Ala Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp

275 280 285 275 280 285

Leu Val Asn Gly Gly Ala Glu Pro Gln Pro Lys Glu Asn Gly Leu ProLeu Val Asn Gly Gly Ala Glu Pro Gln Pro Lys Glu Asn Gly Leu Pro

290 295 300 290 295 300

Leu Glu Gly Leu Leu Gln Ser Ile Ile Asn Lys Lys Pro Val Leu IleLeu Glu Gly Leu Leu Gln Ser Ile Ile Asn Lys Lys Pro Val Leu Ile

305 310 315 320305 310 315 320

Pro Ile Ala Gly Asn Asn Ala Ser His Ile Asp Ile Glu Arg Glu ThrPro Ile Ala Gly Asn Asn Ala Ser His Ile Asp Ile Glu Arg Glu Thr

325 330 335 325 330 335

His Phe Asp Gly Thr Asn Tyr Leu Ala Ile Lys Pro Gly Val Val IleHis Phe Asp Gly Thr Asn Tyr Leu Ala Ile Lys Pro Gly Val Val Ile

340 345 350 340 345 350

Gly Tyr Ala Arg Asn Glu Lys Thr Asn Ala Ala Leu Ala Ala Ala GlyGly Tyr Ala Arg Asn Glu Lys Thr Asn Ala Ala Leu Ala Ala Ala Gly

355 360 365 355 360 365

Ile Lys Val Leu Pro Phe His Gly Asn Gln Leu Ser Leu Gly Met GlyIle Lys Val Leu Pro Phe His Gly Asn Gln Leu Ser Leu Gly Met Gly

370 375 380 370 375 380

Asn Ala Arg Cys Met Ser Met ProAsn Ala Arg Cys Met Ser Met Pro

385 390385 390

<210> 4<210> 4

<211> 410<211> 410

<212> PRT<212> PRT

<213> 精氨酸支原体（Mycoplasma arginini）<213> Mycoplasma arginini

<400> 4<400> 4

Met Ser Val Phe Asp Ser Lys Phe Lys Gly Ile His Val Tyr Ser GluMet Ser Val Phe Asp Ser Lys Phe Lys Gly Ile His Val Tyr Ser Glu

1 5 10 151 5 10 15

Ile Gly Glu Leu Glu Ser Val Leu Val His Glu Pro Gly Arg Glu IleIle Gly Glu Leu Glu Ser Val Leu Val His Glu Pro Gly Arg Glu Ile

20 25 30 20 25 30

Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala IleAsp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile

35 40 45 35 40 45

Leu Glu Ser His Asp Ala Arg Lys Glu His Lys Gln Phe Val Ala GluLeu Glu Ser His Asp Ala Arg Lys Glu His Lys Gln Phe Val Ala Glu

50 55 60 50 55 60

Leu Lys Ala Asn Asp Ile Asn Val Val Glu Leu Ile Asp Leu Val AlaLeu Lys Ala Asn Asp Ile Asn Val Val Glu Leu Ile Asp Leu Val Ala

65 70 75 8065 70 75 80

Glu Thr Tyr Asp Leu Ala Ser Gln Glu Ala Lys Asp Lys Leu Ile GluGlu Thr Tyr Asp Leu Ala Ser Gln Glu Ala Lys Asp Lys Leu Ile Glu

85 90 95 85 90 95

Glu Phe Leu Glu Asp Ser Glu Pro Val Leu Ser Glu Glu His Lys ValGlu Phe Leu Glu Asp Ser Glu Pro Val Leu Ser Glu Glu His Lys Val

100 105 110 100 105 110

Val Val Arg Asn Phe Leu Lys Ala Lys Lys Thr Ser Arg Glu Leu ValVal Val Arg Asn Phe Leu Lys Ala Lys Lys Thr Ser Arg Glu Leu Val

115 120 125 115 120 125

Glu Ile Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Glu AlaGlu Ile Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Glu Ala

130 135 140 130 135 140

Asp His Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr ArgAsp His Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg

145 150 155 160145 150 155 160

Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met ArgAsp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg

165 170 175 165 170 175

Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Val Phe SerTyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Val Phe Ser

180 185 190 180 185 190

Asn His Pro Lys Leu Ile Asn Thr Pro Trp Tyr Tyr Asp Pro Ser LeuAsn His Pro Lys Leu Ile Asn Thr Pro Trp Tyr Tyr Asp Pro Ser Leu

195 200 205 195 200 205

Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp ThrLys Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr

210 215 220 210 215 220

Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Gln Thr Val Thr LeuLeu Val Val Gly Val Ser Glu Arg Thr Asp Leu Gln Thr Val Thr Leu

225 230 235 240225 230 235 240

Leu Ala Lys Asn Ile Val Ala Asn Lys Glu Cys Glu Phe Lys Arg IleLeu Ala Lys Asn Ile Val Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile

245 250 255 245 250 255

Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp ThrVal Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr

260 265 270 260 265 270

Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile AlaTrp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala

275 280 285 275 280 285

Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly AlaAsn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ala

290 295 300 290 295 300

Glu Pro Gln Pro Val Glu Asn Gly Leu Pro Leu Glu Gly Leu Leu GlnGlu Pro Gln Pro Val Glu Asn Gly Leu Pro Leu Glu Gly Leu Leu Gln

305 310 315 320305 310 315 320

Ser Ile Ile Asn Lys Lys Pro Val Leu Ile Pro Ile Ala Gly Glu GlySer Ile Ile Asn Lys Lys Pro Val Leu Ile Pro Ile Ala Gly Glu Gly

325 330 335 325 330 335

Ala Ser Gln Met Glu Ile Glu Arg Glu Thr His Phe Asp Gly Thr AsnAla Ser Gln Met Glu Ile Glu Arg Glu Thr His Phe Asp Gly Thr Asn

340 345 350 340 345 350

Tyr Leu Ala Ile Arg Pro Gly Val Val Ile Gly Tyr Ser Arg Asn GluTyr Leu Ala Ile Arg Pro Gly Val Val Ile Gly Tyr Ser Arg Asn Glu

355 360 365 355 360 365

Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Lys Val Leu Pro PheLys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Lys Val Leu Pro Phe

370 375 380 370 375 380

His Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met SerHis Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser

385 390 395 400385 390 395 400

Met Pro Leu Ser Arg Lys Asp Val Lys TrpMet Pro Leu Ser Arg Lys Asp Val Lys Trp

405 410 405 410

<210> 5<210> 5

<211> 409<211> 409

<212> PRT<212> PRT

<213> 关节炎支原体（Mycoplasma arthritidis）<213> Mycoplasma arthritidis

<400> 5<400> 5

Met Ser Val Phe Asp Ser Lys Phe Lys Gly Ile His Val Tyr Ser GluMet Ser Val Phe Asp Ser Lys Phe Lys Gly Ile His Val Tyr Ser Glu

1 5 10 151 5 10 15

Ile Gly Glu Leu Glu Thr Val Leu Val His Glu Pro Gly Lys Glu IleIle Gly Glu Leu Glu Thr Val Leu Val His Glu Pro Gly Lys Glu Ile

20 25 30 20 25 30

Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala IleAsp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile

35 40 45 35 40 45

Leu Glu Ser His Asp Ala Arg Lys Glu His Lys Glu Phe Val Ala GluLeu Glu Ser His Asp Ala Arg Lys Glu His Lys Glu Phe Val Ala Glu

50 55 60 50 55 60

Leu Lys Lys Arg Gly Ile Asn Val Val Glu Leu Val Asp Leu Ile ValLeu Lys Lys Arg Gly Ile Asn Val Val Glu Leu Val Asp Leu Ile Val

65 70 75 8065 70 75 80

Glu Thr Tyr Asp Leu Ala Ser Lys Glu Ala Lys Glu Lys Leu Leu GluGlu Thr Tyr Asp Leu Ala Ser Lys Glu Ala Lys Glu Lys Leu Leu Glu

85 90 95 85 90 95

Glu Phe Leu Asp Asp Ser Val Pro Val Leu Ser Asp Glu His Arg AlaGlu Phe Leu Asp Asp Ser Val Pro Val Leu Ser Asp Glu His Arg Ala

100 105 110 100 105 110

Ala Val Lys Lys Phe Leu Gln Ser Gln Lys Ser Thr Arg Ser Leu ValAla Val Lys Lys Phe Leu Gln Ser Gln Lys Ser Thr Arg Ser Leu Val

115 120 125 115 120 125

Glu Tyr Met Ile Ala Gly Ile Thr Lys His Asp Leu Lys Ile Glu SerGlu Tyr Met Ile Ala Gly Ile Thr Lys His Asp Leu Lys Ile Glu Ser

130 135 140 130 135 140

Asp Leu Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr ArgAsp Leu Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg

145 150 155 160145 150 155 160

Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met ArgAsp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg

165 170 175 165 170 175

Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Val Phe SerTyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Val Phe Ser

180 185 190 180 185 190

Asn His Pro Lys Leu Val Asn Thr Pro Trp Tyr Tyr Asp Pro Ala GluAsn His Pro Lys Leu Val Asn Thr Pro Trp Tyr Tyr Asp Pro Ala Glu

195 200 205 195 200 205

Gly Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp ThrGly Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr

210 215 220 210 215 220

Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Gln Thr Ile Thr LeuLeu Val Val Gly Val Ser Glu Arg Thr Asp Leu Gln Thr Ile Thr Leu

225 230 235 240225 230 235 240

Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu Cys Glu Phe Lys Arg IleLeu Ala Lys Asn Ile Lys Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile

245 250 255 245 250 255

Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp ThrVal Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr

260 265 270 260 265 270

Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile AlaTrp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala

275 280 285 275 280 285

Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly AspAsn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Asp

290 295 300 290 295 300

Ala Pro Gln Pro Val Asp Asn Gly Leu Pro Leu Glu Asp Leu Leu LysAla Pro Gln Pro Val Asp Asn Gly Leu Pro Leu Glu Asp Leu Leu Lys

305 310 315 320305 310 315 320

Ser Ile Ile Gly Lys Lys Pro Thr Leu Ile Pro Ile Ala Gly Ala GlySer Ile Ile Gly Lys Lys Pro Thr Leu Ile Pro Ile Ala Gly Ala Gly

325 330 335 325 330 335

Ala Ser Gln Ile Asp Ile Glu Arg Glu Thr His Phe Asp Gly Thr AsnAla Ser Gln Ile Asp Ile Glu Arg Glu Thr His Phe Asp Gly Thr Asn

340 345 350 340 345 350

Tyr Leu Ala Val Ala Pro Gly Ile Val Ile Gly Tyr Ala Arg Asn GluTyr Leu Ala Val Ala Pro Gly Ile Val Ile Gly Tyr Ala Arg Asn Glu

355 360 365 355 360 365

Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Thr Val Leu Pro PheLys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Thr Val Leu Pro Phe

370 375 380 370 375 380

Arg Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met SerArg Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser

385 390 395 400385 390 395 400

Met Pro Leu Ser Arg Lys Asp Val LysMet Pro Leu Ser Arg Lys Asp Val Lys

405 405

<210> 6<210> 6

<211> 408<211> 408

<212> PRT<212> PRT

<213> 口腔支原体（Mycoplasma orale）<213> Mycoplasma orale

<220><220>

<221> misc_feature<221> misc_feature

<222> (201)..(201)<222> (201)..(201)

<223> Xaa可以是任何天然存在的氨基酸<223> Xaa can be any naturally occurring amino acid

<220><220>

<221> misc_feature<221> misc_feature

<222> (263)..(263)<222> (263)..(263)

<223> Xaa可以是任何天然存在的氨基酸<223> Xaa can be any naturally occurring amino acid

<220><220>

<221> misc_feature<221> misc_feature

<222> (272)..(272)<222> (272)..(272)

<223> Xaa可以是任何天然存在的氨基酸<223> Xaa can be any naturally occurring amino acid

<220><220>

<221> misc_feature<221> misc_feature

<222> (294)..(294)<222> (294)..(294)

<223> Xaa可以是任何天然存在的氨基酸<223> Xaa can be any naturally occurring amino acid

<400> 6<400> 6

Ser Val Phe Ser Asp Lys Phe Asn Gly Ile His Val Tyr Ser Glu IleSer Val Phe Ser Asp Lys Phe Asn Gly Ile His Val Tyr Ser Glu Ile

1 5 10 151 5 10 15

Gly Asp Leu Glu Ser Val Leu Val His Glu Pro Gly Lys Glu Ile AspGly Asp Leu Glu Ser Val Leu Val His Glu Pro Gly Lys Glu Ile Asp

20 25 30 20 25 30

Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile LeuTyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu

35 40 45 35 40 45

Glu Ser Thr Asp Ala Arg Lys Glu His Lys Glu Phe Val Glu Ile LeuGlu Ser Thr Asp Ala Arg Lys Glu His Lys Glu Phe Val Glu Ile Leu

50 55 60 50 55 60

Lys Lys Gln Gly Ile Asn Val Val Glu Leu Val Asp Leu Val Val GluLys Lys Gln Gly Ile Asn Val Val Glu Leu Val Asp Leu Val Val Glu

65 70 75 8065 70 75 80

Thr Tyr Asn Leu Val Asp Lys Lys Thr Gln Glu Lys Leu Leu Lys AspThr Tyr Asn Leu Val Asp Lys Lys Thr Gln Glu Lys Leu Leu Lys Asp

85 90 95 85 90 95

Phe Leu Asp Asp Ser Glu Pro Val Leu Ser Pro Glu His Arg Lys AlaPhe Leu Asp Asp Ser Glu Pro Val Leu Ser Pro Glu His Arg Lys Ala

100 105 110 100 105 110

Val Glu Lys Phe Leu Lys Ser Leu Lys Ser Thr Lys Glu Leu Ile GlnVal Glu Lys Phe Leu Lys Ser Leu Lys Ser Thr Lys Glu Leu Ile Gln

115 120 125 115 120 125

Tyr Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Lys Ala AspTyr Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Lys Ala Asp

130 135 140 130 135 140

Lys Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg AspLys Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp

145 150 155 160145 150 155 160

Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg TyrPro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg Tyr

165 170 175 165 170 175

Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Lys Phe Ile Phe Thr AsnLys Val Arg Gln Arg Glu Thr Leu Phe Ser Lys Phe Ile Phe Thr Asn

180 185 190 180 185 190

His Pro Lys Leu Val Lys Thr Pro Xaa Tyr Tyr Asp Pro Ala Met LysHis Pro Lys Leu Val Lys Thr Pro Xaa Tyr Tyr Asp Pro Ala Met Lys

195 200 205 195 200 205

Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr LeuLeu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr Leu

210 215 220 210 215 220

Val Val Gly Val Ser Glu Arg Thr Asp Leu Glu Thr Ile Thr Leu LeuVal Val Gly Val Ser Glu Arg Thr Asp Leu Glu Thr Ile Thr Leu Leu

225 230 235 240225 230 235 240

Ala Lys Asn Ile Lys Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile ValAla Lys Asn Ile Lys Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile Val

245 250 255 245 250 255

Ala Ile Asn Val Pro Lys Xaa Thr Asn Leu Met His Leu Asp Thr XaaAla Ile Asn Val Pro Lys Xaa Thr Asn Leu Met His Leu Asp Thr Xaa

260 265 270 260 265 270

Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala AsnLeu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala Asn

275 280 285 275 280 285

Asp Val Phe Lys Phe Xaa Asp Tyr Asp Leu Val Asn Gly Gly Ser AsnAsp Val Phe Lys Phe Xaa Asp Tyr Asp Leu Val Asn Gly Gly Ser Asn

290 295 300 290 295 300

Pro Glu Pro Val Val Asn Gly Leu Pro Leu Asp Lys Leu Leu Glu SerPro Glu Pro Val Val Asn Gly Leu Pro Leu Asp Lys Leu Leu Glu Ser

305 310 315 320305 310 315 320

Ile Ile Asn Lys Lys Pro Val Leu Ile Pro Ile Ala Gly Lys Gly AlaIle Ile Asn Lys Lys Pro Val Leu Ile Pro Ile Ala Gly Lys Gly Ala

325 330 335 325 330 335

Thr Glu Ile Glu Thr Ala Val Glu Thr His Phe Asp Gly Thr Asn TyrThr Glu Ile Glu Thr Ala Val Glu Thr His Phe Asp Gly Thr Asn Tyr

340 345 350 340 345 350

Leu Ala Ile Lys Pro Gly Val Val Val Gly Tyr Ser Arg Asn Val LysLeu Ala Ile Lys Pro Gly Val Val Val Gly Tyr Ser Arg Asn Val Lys

355 360 365 355 360 365

Thr Asn Ala Ala Leu Glu Ala Asn Gly Ile Lys Val Leu Pro Phe LysThr Asn Ala Ala Leu Glu Ala Asn Gly Ile Lys Val Leu Pro Phe Lys

370 375 380 370 375 380

Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser MetGly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser Met

385 390 395 400385 390 395 400

Pro Leu Ser Arg Lys Asp Val LysPro Leu Ser Arg Lys Asp Val Lys

405 405

<210> 7<210> 7

<211> 391<211> 391

<212> PRT<212> PRT

<213> 猫支原体（Mycoplasma gateae）<213> Mycoplasma gateae

<400> 7<400> 7

Ile His Val Tyr Ser Glu Ile Gly Glu Leu Glu Ser Val Leu Val HisIle His Val Tyr Ser Glu Ile Gly Glu Leu Glu Ser Val Leu Val His

1 5 10 151 5 10 15

Glu Pro Gly Arg Glu Ile Asp Tyr Ile Thr Pro Ala Arg Leu Asp GluGlu Pro Gly Arg Glu Ile Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu

20 25 30 20 25 30

Leu Leu Phe Ser Ala Ile Leu Glu Ser His Asp Ala Arg Lys Glu HisLeu Leu Phe Ser Ala Ile Leu Glu Ser His Asp Ala Arg Lys Glu His

35 40 45 35 40 45

Lys Leu Phe Val Ser Glu Leu Lys Ala Asn Asp Ile Asn Val Val GluLys Leu Phe Val Ser Glu Leu Lys Ala Asn Asp Ile Asn Val Val Glu

50 55 60 50 55 60

Leu Thr Asp Leu Val Thr Glu Thr Tyr Asp Leu Ala Ser Gln Glu AlaLeu Thr Asp Leu Val Thr Glu Thr Tyr Asp Leu Ala Ser Gln Glu Ala

65 70 75 8065 70 75 80

Lys Asp Asn Leu Ile Glu Glu Phe Leu Glu Asp Ser Glu Pro Val LeuLys Asp Asn Leu Ile Glu Glu Phe Leu Glu Asp Ser Glu Pro Val Leu

85 90 95 85 90 95

Thr Glu Glu Leu Lys Ser Val Val Arg Thr Tyr Leu Lys Ser Ile LysThr Glu Glu Leu Lys Ser Val Val Arg Thr Tyr Leu Lys Ser Ile Lys

100 105 110 100 105 110

Ser Thr Arg Glu Leu Ile Gln Met Met Met Ala Gly Ile Thr Lys TyrSer Thr Arg Glu Leu Ile Gln Met Met Met Ala Gly Ile Thr Lys Tyr

115 120 125 115 120 125

Asp Leu Gly Ile Glu Ala Asp His Glu Leu Ile Val Asp Pro Met ProAsp Leu Gly Ile Glu Ala Asp His Glu Leu Ile Val Asp Pro Met Pro

130 135 140 130 135 140

Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser Val Gly Asn Gly ValAsn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser Val Gly Asn Gly Val

145 150 155 160145 150 155 160

Thr Ile His Tyr Met Arg Tyr Lys Val Arg Gln Arg Glu Thr Leu PheThr Ile His Tyr Met Arg Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe

165 170 175 165 170 175

Ser Arg Phe Val Phe Ser Asn His Pro Lys Leu Val Asn Thr Pro TrpSer Arg Phe Val Phe Ser Asn His Pro Lys Leu Val Asn Thr Pro Trp

180 185 190 180 185 190

Tyr Tyr Asp Pro Ser Leu Lys Leu Ser Ile Glu Gly Gly Asp Val PheTyr Tyr Asp Pro Ser Leu Lys Leu Ser Ile Glu Gly Gly Asp Val Phe

195 200 205 195 200 205

Ile Tyr Asn Asn Asn Thr Leu Val Val Gly Val Ser Glu Arg Thr AspIle Tyr Asn Asn Asn Thr Leu Val Val Gly Val Ser Glu Arg Thr Asp

210 215 220 210 215 220

Leu Glu Thr Val Thr Leu Leu Ala Lys Asn Ile Val Ala Asn Lys GluLeu Glu Thr Val Thr Leu Leu Ala Lys Asn Ile Val Ala Asn Lys Glu

225 230 235 240225 230 235 240

Cys Glu Phe Lys Arg Ile Val Ala Ile Asn Val Pro Lys Trp Thr AsnCys Glu Phe Lys Arg Ile Val Ala Ile Asn Val Pro Lys Trp Thr Asn

245 250 255 245 250 255

Leu Met His Leu Asp Thr Trp Leu Thr Met Leu Asp Lys Asp Lys PheLeu Met His Leu Asp Thr Trp Leu Thr Met Leu Asp Lys Asp Lys Phe

260 265 270 260 265 270

Leu Tyr Ser Pro Ile Ala Asn Asp Val Phe Lys Phe Trp Asp Tyr AspLeu Tyr Ser Pro Ile Ala Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp

275 280 285 275 280 285

Leu Val Asn Gly Gly Glu Glu Pro Gln Pro Val Glu Asn Gly Leu ProLeu Val Asn Gly Gly Glu Glu Pro Gln Pro Val Glu Asn Gly Leu Pro

290 295 300 290 295 300

Leu Glu Gly Leu Leu Glu Ser Ile Ile Asn Lys Lys Pro Ile Leu IleLeu Glu Gly Leu Leu Glu Ser Ile Ile Asn Lys Lys Pro Ile Leu Ile

305 310 315 320305 310 315 320

Pro Ile Ala Gly Glu Gly Ala Ser Gln Ile Asp Ile Glu Arg Glu ThrPro Ile Ala Gly Glu Gly Ala Ser Gln Ile Asp Ile Glu Arg Glu Thr

325 330 335 325 330 335

His Phe Asp Gly Thr Asn Tyr Leu Ala Ile Arg Pro Gly Val Val IleHis Phe Asp Gly Thr Asn Tyr Leu Ala Ile Arg Pro Gly Val Val Ile

340 345 350 340 345 350

Gly Tyr Ser Arg Asn Glu Lys Thr Asn Ala Ala Leu Glu Ala Ala GlyGly Tyr Ser Arg Asn Glu Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly

355 360 365 355 360 365

Ile Lys Val Leu Pro Phe His Gly Asn Gln Leu Ser Leu Gly Met GlyIle Lys Val Leu Pro Phe His Gly Asn Gln Leu Ser Leu Gly Met Gly

370 375 380 370 375 380

Asn Ala Arg Cys Met Ser MetAsn Ala Arg Cys Met Ser Met

385 390385 390

<210> 8<210> 8

<211> 392<211> 392

<212> PRT<212> PRT

<213> 海豹支原体（Mycoplasma phocidae）<213> Mycoplasma phocidae

<400> 8<400> 8

Ile His Val Tyr Ser Glu Ile Gly Glu Leu Gln Thr Val Leu Val HisIle His Val Tyr Ser Glu Ile Gly Glu Leu Gln Thr Val Leu Val His

1 5 10 151 5 10 15

Glu Pro Gly Arg Glu Ile Glu Tyr Ile Thr Pro Ala Arg Leu Asp GluGlu Pro Gly Arg Glu Ile Glu Tyr Ile Thr Pro Ala Arg Leu Asp Glu

20 25 30 20 25 30

Leu Leu Phe Ser Ala Ile Leu Glu Ser His Asp Ala Arg Lys Glu HisLeu Leu Phe Ser Ala Ile Leu Glu Ser His Asp Ala Arg Lys Glu His

35 40 45 35 40 45

Gln Glu Phe Val Ala Glu Leu Lys Lys Asn Asn Ile Asn Val Val GluGln Glu Phe Val Ala Glu Leu Lys Lys Asn Asn Ile Asn Val Val Glu

50 55 60 50 55 60

Leu Thr Asp Leu Val Ser Glu Thr Tyr Asp Met Val Ser Lys Glu LysLeu Thr Asp Leu Val Ser Glu Thr Tyr Asp Met Val Ser Lys Glu Lys

65 70 75 8065 70 75 80

Gln Glu Lys Leu Ile Glu Glu Phe Leu Glu Asp Ser Glu Pro Val LeuGln Glu Lys Leu Ile Glu Glu Phe Leu Glu Asp Ser Glu Pro Val Leu

85 90 95 85 90 95

Ser Glu Glu His Lys Gly Leu Val Arg Lys Phe Leu Lys Ser Leu LysSer Glu Glu His Lys Gly Leu Val Arg Lys Phe Leu Lys Ser Leu Lys

100 105 110 100 105 110

Ser Ser Lys Glu Leu Ile Gln Tyr Met Met Ala Gly Ile Thr Lys HisSer Ser Lys Glu Leu Ile Gln Tyr Met Met Ala Gly Ile Thr Lys His

115 120 125 115 120 125

Asp Leu Asn Ile Glu Ala Asp His Glu Leu Ile Val Asp Pro Met ProAsp Leu Asn Ile Glu Ala Asp His Glu Leu Ile Val Asp Pro Met Pro

130 135 140 130 135 140

Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser Val Gly Asn Gly ValAsn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser Val Gly Asn Gly Val

145 150 155 160145 150 155 160

Thr Ile His Tyr Met Arg Tyr Lys Val Arg Gln Arg Glu Thr Leu PheThr Ile His Tyr Met Arg Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe

165 170 175 165 170 175

Ser Arg Phe Ile Phe Ala Asn His Pro Lys Leu Met Asn Thr Pro LeuSer Arg Phe Ile Phe Ala Asn His Pro Lys Leu Met Asn Thr Pro Leu

180 185 190 180 185 190

Tyr Tyr Asn Pro Asp Met Lys Leu Ser Ile Glu Gly Gly Asp Val PheTyr Tyr Asn Pro Asp Met Lys Leu Ser Ile Glu Gly Gly Asp Val Phe

195 200 205 195 200 205

Val Tyr Asn Asn Glu Thr Leu Val Val Gly Val Ser Glu Arg Thr AspVal Tyr Asn Asn Glu Thr Leu Val Val Gly Val Ser Glu Arg Thr Asp

210 215 220 210 215 220

Leu Asp Thr Ile Thr Leu Leu Ala Lys Asn Ile Lys Ala Asn Lys GluLeu Asp Thr Ile Thr Leu Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu

225 230 235 240225 230 235 240

Arg Glu Phe Lys Arg Ile Val Ala Ile Asn Val Pro Lys Trp Thr AsnArg Glu Phe Lys Arg Ile Val Ala Ile Asn Val Pro Lys Trp Thr Asn

245 250 255 245 250 255

Leu Met His Leu Asp Thr Trp Leu Thr Met Leu Asp Lys Asp Lys PheLeu Met His Leu Asp Thr Trp Leu Thr Met Leu Asp Lys Asp Lys Phe

260 265 270 260 265 270

Leu Tyr Ser Pro Ile Ala Asn Asp Val Phe Lys Phe Trp Asp Tyr AspLeu Tyr Ser Pro Ile Ala Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp

275 280 285 275 280 285

Leu Val Asn Gly Gly Asp Glu Pro Gln Pro Lys Val Asn Gly Leu ProLeu Val Asn Gly Gly Asp Glu Pro Gln Pro Lys Val Asn Gly Leu Pro

290 295 300 290 295 300

Leu Glu Lys Leu Leu Glu Ser Ile Ile Asn Lys Lys Pro Ile Leu IleLeu Glu Lys Leu Leu Glu Ser Ile Ile Asn Lys Lys Pro Ile Leu Ile

305 310 315 320305 310 315 320

Pro Ile Ala Gly Thr Ser Ala Ser Asn Ile Asp Val Glu Arg Glu ThrPro Ile Ala Gly Thr Ser Ala Ser Asn Ile Asp Val Glu Arg Glu Thr

325 330 335 325 330 335

His Phe Asp Gly Thr Asn Tyr Leu Ala Ile Ala Pro Gly Val Val IleHis Phe Asp Gly Thr Asn Tyr Leu Ala Ile Ala Pro Gly Val Val Ile

340 345 350 340 345 350

Gly Tyr Ser Arg Asn Val Lys Thr Asn Glu Ala Leu Glu Ala Ala GlyGly Tyr Ser Arg Asn Val Lys Thr Asn Glu Ala Leu Glu Ala Ala Gly

355 360 365 355 360 365

Ile Lys Val Leu Pro Phe Lys Gly Asn Gln Leu Ser Leu Gly Met GlyIle Lys Val Leu Pro Phe Lys Gly Asn Gln Leu Ser Leu Gly Met Gly

370 375 380 370 375 380

Asn Ala Arg Cys Met Ser Met ProAsn Ala Arg Cys Met Ser Met Pro

385 390385 390

<210> 9<210> 9

<211> 401<211> 401

<212> PRT<212> PRT

<213> 鸽支原体（Mycoplasma columbinum）<213> Mycoplasma columbinum

<400> 9<400> 9

Met Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Glu Leu Lys Glu ValMet Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Glu Leu Lys Glu Val

1 5 10 151 5 10 15

Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser ArgLeu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser Arg

20 25 30 20 25 30

Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Asn Glu Ala IleLeu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Asn Glu Ala Ile

35 40 45 35 40 45

Lys Glu His Lys Gly Phe Leu Lys Ile Leu Gln Asp Lys Gly Ile LysLys Glu His Lys Gly Phe Leu Lys Ile Leu Gln Asp Lys Gly Ile Lys

50 55 60 50 55 60

Val Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Thr Tyr His AlaVal Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Thr Tyr His Ala

65 70 75 8065 70 75 80

Thr Gln Lys Glu Arg Glu Ala Phe Ile Glu Lys Trp Leu Asp Glu AlaThr Gln Lys Glu Arg Glu Ala Phe Ile Glu Lys Trp Leu Asp Glu Ala

85 90 95 85 90 95

Glu Pro Ala Leu Thr Lys Asp Leu Arg Ala Lys Val Lys Ser Tyr ValGlu Pro Ala Leu Thr Lys Asp Leu Arg Ala Lys Val Lys Ser Tyr Val

100 105 110 100 105 110

Leu Ser Lys Glu Gly Thr Pro Val Ala Met Val Arg Thr Met Met AlaLeu Ser Lys Glu Gly Thr Pro Val Ala Met Val Arg Thr Met Met Ala

115 120 125 115 120 125

Gly Val Ser Lys Gln Glu Leu Asn Val Glu Ser Glu Thr Glu Leu ValGly Val Ser Lys Gln Glu Leu Asn Val Glu Ser Glu Thr Glu Leu Val

130 135 140 130 135 140

Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala SerVal Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser

145 150 155 160145 150 155 160

Ala Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Thr Arg LysAla Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Thr Arg Lys

165 170 175 165 170 175

Arg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ala Thr His Pro Asp TyrArg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ala Thr His Pro Asp Tyr

180 185 190 180 185 190

Lys Thr Thr Pro His Trp Phe Asp Arg Leu Asp Glu Gly Asn Ile GluLys Thr Thr Pro His Trp Phe Asp Arg Leu Asp Glu Gly Asn Ile Glu

195 200 205 195 200 205

Gly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly ValGly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly Val

210 215 220 210 215 220

Ser Glu Arg Thr Asn Lys Glu Ala Ile Leu Thr Ile Ala Lys Lys IleSer Glu Arg Thr Asn Lys Glu Ala Ile Leu Thr Ile Ala Lys Lys Ile

225 230 235 240225 230 235 240

Lys Asn Asn Lys Glu Ala Lys Phe Lys Lys Ile Val Ala Ile Asn ValLys Asn Asn Lys Glu Ala Lys Phe Lys Lys Ile Val Ala Ile Asn Val

245 250 255 245 250 255

Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met ValPro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Val

260 265 270 260 265 270

Asp Lys Asp Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu LysAsp Lys Asp Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu Lys

275 280 285 275 280 285

Val Trp Glu Ile Asp Leu Ser Lys Glu Ile Glu Met Val Glu Thr AsnVal Trp Glu Ile Asp Leu Ser Lys Glu Ile Glu Met Val Glu Thr Asn

290 295 300 290 295 300

Lys Pro Leu Ala Asp Val Leu Glu Ser Ile Ile Gly Val Lys Pro ValLys Pro Leu Ala Asp Val Leu Glu Ser Ile Ile Gly Val Lys Pro Val

305 310 315 320305 310 315 320

Leu Ile Pro Ile Ala Gly Lys Gly Ala Thr Gln Leu Asp Ile Asp IleLeu Ile Pro Ile Ala Gly Lys Gly Ala Thr Gln Leu Asp Ile Asp Ile

325 330 335 325 330 335

Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly ValGlu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly Val

340 345 350 340 345 350

Val Val Gly Tyr Ser Arg Asn Ile Lys Thr Glu Ala Ala Leu Arg AlaVal Val Gly Tyr Ser Arg Asn Ile Lys Thr Glu Ala Ala Leu Arg Ala

355 360 365 355 360 365

Ala Gly Val Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu GlyAla Gly Val Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu Gly

370 375 380 370 375 380

Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp ValMet Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp Val

385 390 395 400385 390 395 400

LysLys

<210> 10<210> 10

<211> 407<211> 407

<212> PRT<212> PRT

<213> 衣阿华支原体（Mycoplasma iowae）<213> Mycoplasma iowae

<400> 10<400> 10

Met Gly Asn Asn Ile Pro Lys Lys Ile Asn Val Phe Ser Glu Ile GlyMet Gly Asn Asn Ile Pro Lys Lys Ile Asn Val Phe Ser Glu Ile Gly

1 5 10 151 5 10 15

Asn Leu Lys Arg Val Leu Val His Thr Pro Gly Lys Glu Ile Glu TyrAsn Leu Lys Arg Val Leu Val His Thr Pro Gly Lys Glu Ile Glu Tyr

20 25 30 20 25 30

Val Thr Pro Gln Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu AspVal Thr Pro Gln Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Asp

35 40 45 35 40 45

Pro Val Arg Ala Arg Glu Glu His Lys Glu Phe Ile Lys Ile Leu GluPro Val Arg Ala Arg Glu Glu His Lys Glu Phe Ile Lys Ile Leu Glu

50 55 60 50 55 60

Ser Gln Gly Val Glu Val Val Gln Leu Val Asp Leu Thr Ala Glu ThrSer Gln Gly Val Glu Val Val Gln Leu Val Asp Leu Thr Ala Glu Thr

65 70 75 8065 70 75 80

Tyr Asp Val Ala Glu Ser Gln Ala Lys Glu Asn Phe Ile Gln Lys TrpTyr Asp Val Ala Glu Ser Gln Ala Lys Glu Asn Phe Ile Gln Lys Trp

85 90 95 85 90 95

Leu Asp Glu Ser Leu Pro Lys Leu Thr Asp Glu Asn Arg Asn Lys ValLeu Asp Glu Ser Leu Pro Lys Leu Thr Asp Glu Asn Arg Asn Lys Val

100 105 110 100 105 110

Tyr Ser Leu Leu Lys Ser Leu Glu Lys Asp Pro Lys Glu Met Ile ArgTyr Ser Leu Leu Lys Ser Leu Glu Lys Asp Pro Lys Glu Met Ile Arg

115 120 125 115 120 125

Lys Met Met Ser Gly Val Leu Ala Ser Glu Ile Gly Val Lys Ser AspLys Met Met Ser Gly Val Leu Ala Ser Glu Ile Gly Val Lys Ser Asp

130 135 140 130 135 140

Val Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg AspVal Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp

145 150 155 160145 150 155 160

Pro Phe Ala Ser Val Gly Asn Gly Ile Thr Leu His Arg Met Phe ArgPro Phe Ala Ser Val Gly Asn Gly Ile Thr Leu His Arg Met Phe Arg

165 170 175 165 170 175

Pro Thr Arg Arg Arg Glu Thr Ile Phe Ala Asp Phe Ile Phe Ser AsnPro Thr Arg Arg Arg Glu Thr Ile Phe Ala Asp Phe Ile Phe Ser Asn

180 185 190 180 185 190

His Pro Glu Tyr Lys Ser Thr Gln Lys Tyr Tyr Glu Arg Glu Asp LysHis Pro Glu Tyr Lys Ser Thr Gln Lys Tyr Tyr Glu Arg Glu Asp Lys

195 200 205 195 200 205

Phe Ser Leu Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Lys Thr LeuPhe Ser Leu Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Lys Thr Leu

210 215 220 210 215 220

Val Val Gly Val Ser Glu Arg Thr Glu Lys Gly Ala Ile Lys Ala LeuVal Val Gly Val Ser Glu Arg Thr Glu Lys Gly Ala Ile Lys Ala Leu

225 230 235 240225 230 235 240

Ala Lys Ala Val Gln Asn Asn Ser Asn Met Ser Phe Glu Lys Ile TyrAla Lys Ala Val Gln Asn Asn Ser Asn Met Ser Phe Glu Lys Ile Tyr

245 250 255 245 250 255

Ala Ile Asn Val Pro Lys Met Ser Asn Leu Met His Leu Asp Thr TrpAla Ile Asn Val Pro Lys Met Ser Asn Leu Met His Leu Asp Thr Trp

260 265 270 260 265 270

Leu Thr Met Leu Asp Thr Asp Lys Phe Leu Tyr Ser Pro Asn Met MetLeu Thr Met Leu Asp Thr Asp Lys Phe Leu Tyr Ser Pro Asn Met Met

275 280 285 275 280 285

Gly Val Leu Lys Ile Trp Glu Ile Asp Leu Ser Asp Lys Ser Leu LysGly Val Leu Lys Ile Trp Glu Ile Asp Leu Ser Asp Lys Ser Leu Lys

290 295 300 290 295 300

Trp Lys Glu Ile Arg Asp Ser Leu Asp His Phe Leu Ser Thr Ile IleTrp Lys Glu Ile Arg Asp Ser Leu Asp His Phe Leu Ser Thr Ile Ile

305 310 315 320305 310 315 320

Gly Lys Lys Ala Ile Thr Val Pro Val Ala Gly Lys Asp Ala Met GlnGly Lys Lys Ala Ile Thr Val Pro Val Ala Gly Lys Asp Ala Met Gln

325 330 335 325 330 335

Phe Glu Ile Asp Ile Glu Thr His Phe Asp Ala Thr Asn Phe Ile AlaPhe Glu Ile Asp Ile Glu Thr His Phe Asp Ala Thr Asn Phe Ile Ala

340 345 350 340 345 350

Val Ala Pro Gly Val Val Ile Gly Tyr Asp Arg Asn Lys Lys Thr AsnVal Ala Pro Gly Val Val Ile Gly Tyr Asp Arg Asn Lys Lys Thr Asn

355 360 365 355 360 365

Glu Ala Leu Lys Glu Ala Gly Ile Lys Val Leu Ser Trp Asn Gly AspGlu Ala Leu Lys Glu Ala Gly Ile Lys Val Leu Ser Trp Asn Gly Asp

370 375 380 370 375 380

Gln Leu Ser Leu Gly Met Gly Ser Ala Arg Cys Met Thr Met Pro LeuGln Leu Ser Leu Gly Met Gly Ser Ala Arg Cys Met Thr Met Pro Leu

385 390 395 400385 390 395 400

Tyr Arg Glu Glu Leu Lys LysTyr Arg Glu Glu Leu Lys Lys

405 405

<210> 11<210> 11

<211> 402<211> 402

<212> PRT<212> PRT

<213> 鳄鱼支原体（Mycoplasma crocodyli）<213> Mycoplasma crocodyli

<400> 11<400> 11

Met Asn Lys Ile Asn Val Tyr Ser Glu Val Gly Lys Leu Lys Glu ValMet Asn Lys Ile Asn Val Tyr Ser Glu Val Gly Lys Leu Lys Glu Val

1 5 10 151 5 10 15

Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser ArgLeu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser Arg

20 25 30 20 25 30

Leu Glu Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Asp Ser Ala IleLeu Glu Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Asp Ser Ala Ile

35 40 45 35 40 45

Glu Glu His Lys Arg Phe Leu Lys Ile Leu Glu Asp Asn Asn Ile LysGlu Glu His Lys Arg Phe Leu Lys Ile Leu Glu Asp Asn Asn Ile Lys

50 55 60 50 55 60

Val Ile Gln Leu Asp Gln Leu Val Ala Asp Thr Tyr Glu Leu Val AsnVal Ile Gln Leu Asp Gln Leu Val Ala Asp Thr Tyr Glu Leu Val Asn

65 70 75 8065 70 75 80

Pro Ser Val Arg Asp Ala Phe Ile Glu Lys Trp Leu Asn Glu Ser GluPro Ser Val Arg Asp Ala Phe Ile Glu Lys Trp Leu Asn Glu Ser Glu

85 90 95 85 90 95

Pro Lys Leu Asp Lys Lys Leu Arg Glu Lys Val Lys Glu Tyr Leu LeuPro Lys Leu Asp Lys Lys Lys Leu Arg Glu Lys Val Lys Glu Tyr Leu Leu

100 105 110 100 105 110

His Thr Gln Lys Thr Val Gly Thr Lys Arg Met Val Arg Ile Met MetHis Thr Gln Lys Thr Val Gly Thr Lys Arg Met Val Arg Ile Met Met

115 120 125 115 120 125

Ala Gly Val Asp Arg Val Glu Leu Gly Val Glu Leu Asp Arg Gln LeuAla Gly Val Asp Arg Val Glu Leu Gly Val Glu Leu Asp Arg Gln Leu

130 135 140 130 135 140

Val Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe AlaVal Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala

145 150 155 160145 150 155 160

Ser Ala Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Thr ArgSer Ala Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Thr Arg

165 170 175 165 170 175

Lys Arg Glu Thr Ile Phe Ser Glu Phe Ile Phe Glu Asn His Pro AspLys Arg Glu Thr Ile Phe Ser Glu Phe Ile Phe Glu Asn His Pro Asp

180 185 190 180 185 190

Tyr Lys Thr Thr Pro His Trp Phe Asp Arg Leu Asp Lys Gly Asn IleTyr Lys Thr Thr Pro His Trp Phe Asp Arg Leu Asp Lys Gly Asn Ile

195 200 205 195 200 205

Glu Gly Gly Asp Val Phe Ile Tyr Asn Arg Thr Thr Leu Val Ile GlyGlu Gly Gly Asp Val Phe Ile Tyr Asn Arg Thr Thr Leu Val Ile Gly

210 215 220 210 215 220

Ile Ser Glu Arg Thr Asn Lys Asp Ala Leu Leu Thr Ile Ala Asn AsnIle Ser Glu Arg Thr Asn Lys Asp Ala Leu Leu Thr Ile Ala Asn Asn

225 230 235 240225 230 235 240

Ile Lys Ser Asn Lys Glu Ser Lys Phe Glu Arg Ile Val Ala Val AsnIle Lys Ser Asn Lys Glu Ser Lys Phe Glu Arg Ile Val Ala Val Asn

245 250 255 245 250 255

Val Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr MetVal Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met

260 265 270 260 265 270

Val Asp His Asp Lys Phe Leu Tyr Ser Pro Asn Met Met Lys Thr LeuVal Asp His Asp Lys Phe Leu Tyr Ser Pro Asn Met Met Lys Thr Leu

275 280 285 275 280 285

Lys Phe Trp Thr Ile Asp Leu Thr Lys Pro Ile Lys Met Val Glu LeuLys Phe Trp Thr Ile Asp Leu Thr Lys Pro Ile Lys Met Val Glu Leu

290 295 300 290 295 300

Glu Glu Ser Leu Ser Asp Met Ile Glu Thr Ile Ile Gly Lys Lys ProGlu Glu Ser Leu Ser Asp Met Ile Glu Thr Ile Ile Gly Lys Lys Pro

305 310 315 320305 310 315 320

Val Leu Ile Pro Ile Ala Gly His Asp Ala Ser Pro Leu Asp Val AspVal Leu Ile Pro Ile Ala Gly His Asp Ala Ser Pro Leu Asp Val Asp

325 330 335 325 330 335

Ile Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro GlyIle Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly

340 345 350 340 345 350

Val Val Val Gly Tyr Ser Arg Asn Lys Leu Thr Glu Lys Ala Leu ThrVal Val Val Gly Tyr Ser Arg Asn Lys Leu Thr Glu Lys Ala Leu Thr

355 360 365 355 360 365

Lys Ala Gly Val Lys Val Leu Ser Phe Glu Gly Asn Gln Leu Ser LeuLys Ala Gly Val Lys Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu

370 375 380 370 375 380

Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu AspGly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp

385 390 395 400385 390 395 400

Ile LysIle Lys

<210> 12<210> 12

<211> 430<211> 430

<212> PRT<212> PRT

<213> 发酵支原体（Mycoplasma fermentans）<213> Mycoplasma fermentans

<400> 12<400> 12

Met Gln Ile Ile Ala Lys Ile Asp Leu Leu Thr Asn Met Leu Ile PheMet Gln Ile Ile Ala Lys Ile Asp Leu Leu Thr Asn Met Leu Ile Phe

1 5 10 151 5 10 15

Met Lys Ile Tyr Phe Ile Gly Arg Leu Ile Met Lys Lys Ile Asn ValMet Lys Ile Tyr Phe Ile Gly Arg Leu Ile Met Lys Lys Ile Asn Val

20 25 30 20 25 30

Tyr Ser Glu Tyr Gly Lys Leu Lys Glu Val Leu Val His Thr Pro GlyTyr Ser Glu Tyr Gly Lys Leu Lys Glu Val Leu Val His Thr Pro Gly

35 40 45 35 40 45

Asp Glu Ile Arg Arg Ile Ala Pro Ser Arg Leu Asp Glu Leu Leu PheAsp Glu Ile Arg Arg Ile Ala Pro Ser Arg Leu Asp Glu Leu Leu Phe

50 55 60 50 55 60

Ser Ala Ile Leu Glu Pro Asp Ser Ala Ile Ala Glu His Lys Arg PheSer Ala Ile Leu Glu Pro Asp Ser Ala Ile Ala Glu His Lys Arg Phe

65 70 75 8065 70 75 80

Val Gln Leu Leu Lys Asp Asn Gly Ile Lys Val Ile Gln Leu Asp GluVal Gln Leu Leu Lys Asp Asn Gly Ile Lys Val Ile Gln Leu Asp Glu

85 90 95 85 90 95

Leu Phe Ala Lys Thr Phe Asp Leu Val Ser Glu Ser Val Lys Gln SerLeu Phe Ala Lys Thr Phe Asp Leu Val Ser Glu Ser Val Lys Gln Ser

100 105 110 100 105 110

Leu Ile Glu Arg Trp Leu Asp Glu Cys Glu Pro Lys Leu Asp Ala ThrLeu Ile Glu Arg Trp Leu Asp Glu Cys Glu Pro Lys Leu Asp Ala Thr

115 120 125 115 120 125

Leu Arg Ala Lys Val Lys Glu Tyr Ile Leu Glu Leu Lys Ala Lys SerLeu Arg Ala Lys Val Lys Glu Tyr Ile Leu Glu Leu Lys Ala Lys Ser

130 135 140 130 135 140

Ser Lys Lys Met Val Arg Val Met Met Ala Gly Ile Asp Lys Lys GluSer Lys Lys Met Val Arg Val Met Met Ala Gly Ile Asp Lys Lys Glu

145 150 155 160145 150 155 160

Leu Gly Ile Glu Leu Asp Arg Asp Leu Val Val Asp Pro Met Pro AsnLeu Gly Ile Glu Leu Asp Arg Asp Leu Val Val Asp Pro Met Pro Asn

165 170 175 165 170 175

Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser Val Gly Asn Gly Ile SerLeu Tyr Phe Thr Arg Asp Pro Phe Ala Ser Val Gly Asn Gly Ile Ser

180 185 190 180 185 190

Leu His His Met Lys Tyr Val Thr Arg Gln Arg Glu Thr Ile Phe SerLeu His His Met Lys Tyr Val Thr Arg Gln Arg Glu Thr Ile Phe Ser

195 200 205 195 200 205

Glu Phe Ile Phe Asp Asn Asn Leu Asp Tyr Asn Thr Val Pro Arg TrpGlu Phe Ile Phe Asp Asn Asn Leu Asp Tyr Asn Thr Val Pro Arg Trp

210 215 220 210 215 220

Phe Asp Arg Lys Asp Glu Gly Arg Ile Glu Gly Gly Asp Val Phe IlePhe Asp Arg Lys Asp Glu Gly Arg Ile Glu Gly Gly Asp Val Phe Ile

225 230 235 240225 230 235 240

Tyr Ser Ala Asp Thr Leu Val Val Gly Val Ser Glu Arg Thr Asn LysTyr Ser Ala Asp Thr Leu Val Val Gly Val Ser Glu Arg Thr Asn Lys

245 250 255 245 250 255

Glu Ala Ile Asn Val Met Ala Arg Lys Ile Ala Ala Asp Lys Glu ValGlu Ala Ile Asn Val Met Ala Arg Lys Ile Ala Ala Asp Lys Glu Val

260 265 270 260 265 270

Lys Phe Lys Arg Ile Tyr Ala Ile Asn Val Pro Pro Met Pro Asn LeuLys Phe Lys Arg Ile Tyr Ala Ile Asn Val Pro Pro Met Pro Asn Leu

275 280 285 275 280 285

Met His Leu Asp Thr Trp Leu Thr Met Leu Asp Lys Asn Lys Phe LeuMet His Leu Asp Thr Trp Leu Thr Met Leu Asp Lys Asn Lys Phe Leu

290 295 300 290 295 300

Tyr Ser Pro Asn Met Leu Ser Val Leu Lys Val Trp Arg Ile Asp LeuTyr Ser Pro Asn Met Leu Ser Val Leu Lys Val Trp Arg Ile Asp Leu

305 310 315 320305 310 315 320

Asn Asp Pro Asp Phe Val Trp His Glu Ile Glu Gly Ser Leu Glu GluAsn Asp Pro Asp Phe Val Trp His Glu Ile Glu Gly Ser Leu Glu Glu

325 330 335 325 330 335

Ile Leu Glu Gln Ile Ile Gly Met Lys Pro Ile Leu Ile Pro Ile AlaIle Leu Glu Gln Ile Ile Gly Met Lys Pro Ile Leu Ile Pro Ile Ala

340 345 350 340 345 350

Gly Lys Gly Ala Ser Gln Leu Asp Ile Asp Ile Glu Thr His Phe AspGly Lys Gly Ala Ser Gln Leu Asp Ile Asp Ile Glu Thr His Phe Asp

355 360 365 355 360 365

Gly Thr Asn Tyr Leu Thr Ile Ala Pro Ser Val Val Val Gly Tyr SerGly Thr Asn Tyr Leu Thr Ile Ala Pro Ser Val Val Val Gly Tyr Ser

370 375 380 370 375 380

Arg Asn Glu Lys Thr Glu Lys Ala Leu Lys Ala Ala Lys Val Lys ValArg Asn Glu Lys Thr Glu Lys Ala Leu Lys Ala Ala Lys Val Lys Val

385 390 395 400385 390 395 400

Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu Gly Met Gly Ser Ala ArgLeu Ser Phe Glu Gly Asn Gln Leu Ser Leu Gly Met Gly Ser Ala Arg

405 410 415 405 410 415

Cys Met Ser Met Pro Leu Ile Arg Glu Asp Ile Lys Lys LysCys Met Ser Met Pro Leu Ile Arg Glu Asp Ile Lys Lys Lys

420 425 430 420 425 430

<210> 13<210> 13

<211> 452<211> 452

<212> PRT<212> PRT

<213> 穿透支原体（Mycoplasma penetrans）<213> Mycoplasma penetrans

<400> 13<400> 13

Met Val Ile Thr Ile Ala Leu Asn Ile Leu Asn Lys Ile Tyr Phe LysMet Val Ile Thr Ile Ala Leu Asn Ile Leu Asn Lys Ile Tyr Phe Lys

1 5 10 151 5 10 15

Pro Gln Asn Arg Ser Ile Leu Lys Leu Tyr Arg Leu Pro Ser Leu CysPro Gln Asn Arg Ser Ile Leu Lys Leu Tyr Arg Leu Pro Ser Leu Cys

20 25 30 20 25 30

Thr Gln Ile Ser Ile Phe Ile Gly Gly Lys Met Ser Ser Ile Asp LysThr Gln Ile Ser Ile Phe Ile Gly Gly Lys Met Ser Ser Ile Asp Lys

35 40 45 35 40 45

Asn Ser Leu Gly Asn Gly Ile Asn Val Tyr Ser Glu Ile Gly Glu LeuAsn Ser Leu Gly Asn Gly Ile Asn Val Tyr Ser Glu Ile Gly Glu Leu

50 55 60 50 55 60

Lys Glu Val Leu Val His Thr Pro Gly Asp Glu Ile Arg Tyr Thr AlaLys Glu Val Leu Val His Thr Pro Gly Asp Glu Ile Arg Tyr Thr Ala

65 70 75 8065 70 75 80

Pro Ser Arg Leu Glu Glu Leu Leu Phe Ser Ala Val Leu Lys Ala AspPro Ser Arg Leu Glu Glu Leu Leu Phe Ser Ala Val Leu Lys Ala Asp

85 90 95 85 90 95

Thr Ala Ile Glu Glu His Lys Gly Phe Val Lys Ile Leu Gln Asn AsnThr Ala Ile Glu Glu His Lys Gly Phe Val Lys Ile Leu Gln Asn Asn

100 105 110 100 105 110

Gly Ile Lys Val Ile Gln Leu Cys Asp Leu Val Ala Glu Thr Tyr GluGly Ile Lys Val Ile Gln Leu Cys Asp Leu Val Ala Glu Thr Tyr Glu

115 120 125 115 120 125

Leu Cys Ser Lys Glu Val Arg Asn Ser Phe Ile Glu Gln Tyr Leu AspLeu Cys Ser Lys Glu Val Arg Asn Ser Phe Ile Glu Gln Tyr Leu Asp

130 135 140 130 135 140

Glu Ala Leu Pro Val Leu Lys Lys Glu Ile Arg Pro Val Val Lys AspGlu Ala Leu Pro Val Leu Lys Lys Glu Ile Arg Pro Val Val Lys Asp

145 150 155 160145 150 155 160

Tyr Leu Leu Ser Phe Pro Thr Val Gln Met Val Arg Lys Met Met SerTyr Leu Leu Ser Phe Pro Thr Val Gln Met Val Arg Lys Met Met Ser

165 170 175 165 170 175

Gly Ile Leu Ala Asn Glu Leu Asn Ile Lys Gln Asp Asn Pro Leu IleGly Ile Leu Ala Asn Glu Leu Asn Ile Lys Gln Asp Asn Pro Leu Ile

180 185 190 180 185 190

Ile Asp Gly Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala SerIle Asp Gly Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser

195 200 205 195 200 205

Met Gly Asn Gly Val Ser Ile Asn Cys Met Lys Tyr Pro Thr Arg LysMet Gly Asn Gly Val Ser Ile Asn Cys Met Lys Tyr Pro Thr Arg Lys

210 215 220 210 215 220

Arg Glu Val Ile Phe Ser Arg Phe Val Phe Thr Asn Asn Pro Lys TyrArg Glu Val Ile Phe Ser Arg Phe Val Phe Thr Asn Asn Pro Lys Tyr

225 230 235 240225 230 235 240

Lys Asn Thr Pro Arg Tyr Phe Asp Ile Val Gly Asn Asn Gly Thr IleLys Asn Thr Pro Arg Tyr Phe Asp Ile Val Gly Asn Asn Gly Thr Ile

245 250 255 245 250 255

Glu Gly Gly Asp Ile Phe Ile Tyr Asn Ser Lys Thr Leu Val Ile GlyGlu Gly Gly Asp Ile Phe Ile Tyr Asn Ser Lys Thr Leu Val Ile Gly

260 265 270 260 265 270

Asn Ser Glu Arg Thr Asn Phe Ala Ala Ile Glu Ser Val Ala Lys AsnAsn Ser Glu Arg Thr Asn Phe Ala Ala Ile Glu Ser Val Ala Lys Asn

275 280 285 275 280 285

Ile Gln Ala Asn Lys Asp Cys Thr Phe Glu Arg Ile Val Val Ile AsnIle Gln Ala Asn Lys Asp Cys Thr Phe Glu Arg Ile Val Val Ile Asn

290 295 300 290 295 300

Val Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr MetVal Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met

305 310 315 320305 310 315 320

Leu Asp Tyr Asp Lys Phe Leu Tyr Ser Pro Asn Met Met Asn Val LeuLeu Asp Tyr Asp Lys Phe Leu Tyr Ser Pro Asn Met Met Asn Val Leu

325 330 335 325 330 335

Lys Ile Trp Glu Ile Asp Leu Asn Val Lys Pro Val Lys Phe Val GluLys Ile Trp Glu Ile Asp Leu Asn Val Lys Pro Val Lys Phe Val Glu

340 345 350 340 345 350

Lys Lys Gly Thr Leu Glu Glu Val Leu Tyr Ser Ile Ile Asp Lys LysLys Lys Gly Thr Leu Glu Glu Val Leu Tyr Ser Ile Ile Asp Lys Lys

355 360 365 355 360 365

Pro Ile Leu Ile Pro Ile Ala Gly Lys Gly Ala Asn Gln Leu Asp IlePro Ile Leu Ile Pro Ile Ala Gly Lys Gly Ala Asn Gln Leu Asp Ile

370 375 380 370 375 380

Asp Ile Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala ProAsp Ile Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro

385 390 395 400385 390 395 400

Gly Val Val Val Gly Tyr Glu Arg Asn Glu Lys Thr Gln Lys Ala LeuGly Val Val Val Gly Tyr Glu Arg Asn Glu Lys Thr Gln Lys Ala Leu

405 410 415 405 410 415

Val Glu Ala Gly Ile Lys Val Leu Ser Phe Asn Gly Ser Gln Leu SerVal Glu Ala Gly Ile Lys Val Leu Ser Phe Asn Gly Ser Gln Leu Ser

420 425 430 420 425 430

Leu Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Ile Arg GluLeu Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Ile Arg Glu

435 440 445 435 440 445

Asn Leu Lys LysAsn Leu Lys Lys

450 450

<210> 14<210> 14

<211> 403<211> 403

<212> PRT<212> PRT

<213> 鸡毒支原体（Mycoplasma gallisepticum）<213> Mycoplasma gallisepticum

<400> 14<400> 14

Met Phe Asn Lys Ile Arg Val Tyr Ser Glu Ile Gly Lys Leu Arg LysMet Phe Asn Lys Ile Arg Val Tyr Ser Glu Ile Gly Lys Leu Arg Lys

1 5 10 151 5 10 15

Val Leu Val His Thr Pro Gly Lys Glu Leu Asp Tyr Val Thr Pro GlnVal Leu Val His Thr Pro Gly Lys Glu Leu Asp Tyr Val Thr Pro Gln

20 25 30 20 25 30

Arg Leu Asp Glu Leu Leu Phe Ser Ser Leu Leu Asn Pro Ile Lys AlaArg Leu Asp Glu Leu Leu Phe Ser Ser Leu Leu Asn Pro Ile Lys Ala

35 40 45 35 40 45

Arg Gln Glu His Glu Thr Phe Ile Lys Leu Leu Glu Asp His Asp ValArg Gln Glu His Glu Thr Phe Ile Lys Leu Leu Glu Asp His Asp Val

50 55 60 50 55 60

Glu Cys Val Gln Leu Ser Thr Leu Thr Ala Gln Thr Phe Gln Ala ValGlu Cys Val Gln Leu Ser Thr Leu Thr Ala Gln Thr Phe Gln Ala Val

65 70 75 8065 70 75 80

Asn Ser Lys Ile Gln Glu Glu Phe Ile Asn Arg Trp Leu Asp Glu CysAsn Ser Lys Ile Gln Glu Glu Phe Ile Asn Arg Trp Leu Asp Glu Cys

85 90 95 85 90 95

Leu Pro Val Leu Ser Glu Ile Asn Arg Leu Lys Val Tyr Asp Tyr LeuLeu Pro Val Leu Ser Glu Ile Asn Arg Leu Lys Val Tyr Asp Tyr Leu

100 105 110 100 105 110

Lys Ser Leu Ala Thr Asn Pro Gln Val Met Ile Arg Lys Met Met SerLys Ser Leu Ala Thr Asn Pro Gln Val Met Ile Arg Lys Met Met Ser

115 120 125 115 120 125

Gly Ile Leu Ala Lys Glu Val Gly Ile Gln Ser Glu Val Glu Leu ValGly Ile Leu Ala Lys Glu Val Gly Ile Gln Ser Glu Val Glu Leu Val

130 135 140 130 135 140

Ala Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala SerAla Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser

145 150 155 160145 150 155 160

Ile Gly Lys Gly Ile Thr Leu His Ser Met Phe His Pro Thr Arg LysIle Gly Lys Gly Ile Thr Leu His Ser Met Phe His Pro Thr Arg Lys

165 170 175 165 170 175

Arg Glu Thr Ile Phe Ala Asp Phe Ile Phe Ser His His Pro Glu TyrArg Glu Thr Ile Phe Ala Asp Phe Ile Phe Ser His His Pro Glu Tyr

180 185 190 180 185 190

Lys Asn Ala Pro Lys Tyr Tyr Ser Arg Glu Asp Lys Tyr Ser Ile GluLys Asn Ala Pro Lys Tyr Tyr Ser Arg Glu Asp Lys Tyr Ser Ile Glu

195 200 205 195 200 205

Gly Gly Asp Leu Phe Val Tyr Asp Asp Lys Thr Leu Val Ile Gly ValGly Gly Asp Leu Phe Val Tyr Asp Asp Lys Thr Leu Val Ile Gly Val

210 215 220 210 215 220

Ser Glu Arg Thr Glu Lys Lys Ala Ile Gln Ser Leu Ala Glu Lys LeuSer Glu Arg Thr Glu Lys Lys Ala Ile Gln Ser Leu Ala Glu Lys Leu

225 230 235 240225 230 235 240

Arg Gln Asn Asp Glu Thr Ser Phe Glu Lys Ile Tyr Ala Ile Asn ValArg Gln Asn Asp Glu Thr Ser Phe Glu Lys Ile Tyr Ala Ile Asn Val

245 250 255 245 250 255

Pro Lys Met Ser Asn Leu Met His Leu Asp Thr Trp Leu Thr Met LeuPro Lys Met Ser Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Leu

260 265 270 260 265 270

Asp Tyr Asp Lys Phe Leu Tyr Ser Pro Asn Met Met Gly Val Leu LysAsp Tyr Asp Lys Phe Leu Tyr Ser Pro Asn Met Met Gly Val Leu Lys

275 280 285 275 280 285

Ile Trp Glu Ile Asp Leu Ile His Pro Thr Leu Ile Trp Arg Glu LeuIle Trp Glu Ile Asp Leu Ile His Pro Thr Leu Ile Trp Arg Glu Leu

290 295 300 290 295 300

Asn Glu Ser Leu Glu Gly Phe Leu Ser Met Val Ile Gly Lys Lys AlaAsn Glu Ser Leu Glu Gly Phe Leu Ser Met Val Ile Gly Lys Lys Ala

305 310 315 320305 310 315 320

Thr Leu Ile Pro Val Ala Gly Glu Asp Ser Thr Gln Ile Glu Ile AspThr Leu Ile Pro Val Ala Gly Glu Asp Ser Thr Gln Ile Glu Ile Asp

325 330 335 325 330 335

Val Glu Thr Asn Phe Asp Ala Thr Asn Phe Leu Val Ile Gln Pro GlyVal Glu Thr Asn Phe Asp Ala Thr Asn Phe Leu Val Ile Gln Pro Gly

340 345 350 340 345 350

Val Val Val Gly Tyr Asp Arg Asn Tyr Lys Thr Asn Gln Ala Leu ArgVal Val Val Gly Tyr Asp Arg Asn Tyr Lys Thr Asn Gln Ala Leu Arg

355 360 365 355 360 365

Asp Ala Gly Val Lys Val Ile Ser Trp Asn Gly Asp Gln Leu Ser LeuAsp Ala Gly Val Lys Val Ile Ser Trp Asn Gly Asp Gln Leu Ser Leu

370 375 380 370 375 380

Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Tyr Arg Asp ProGly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Tyr Arg Asp Pro

385 390 395 400385 390 395 400

Ile Lys LysIle Lys Lys

<210> 15<210> 15

<211> 402<211> 402

<212> PRT<212> PRT

<213> 短吻鳄支原体（Mycoplasma alligatoris）<213> Mycoplasma alligatoris

<400> 15<400> 15

Met Ser Lys Ile Asn Val Tyr Ser Glu Val Gly Arg Leu Lys Glu ValMet Ser Lys Ile Asn Val Tyr Ser Glu Val Gly Arg Leu Lys Glu Val

1 5 10 151 5 10 15

Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Thr ArgLeu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Thr Arg

20 25 30 20 25 30

Leu Glu Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Asp Thr Ala IleLeu Glu Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Asp Thr Ala Ile

35 40 45 35 40 45

Glu Glu His Lys Arg Phe Leu Asn Val Leu Glu Lys Asn Gly Ile LysGlu Glu His Lys Arg Phe Leu Asn Val Leu Glu Lys Asn Gly Ile Lys

50 55 60 50 55 60

Ala Ile Gln Leu Asp Glu Leu Val Ala Gln Thr Tyr Asp Gln Val AspAla Ile Gln Leu Asp Glu Leu Val Ala Gln Thr Tyr Asp Gln Val Asp

65 70 75 8065 70 75 80

Gln Lys Ile Lys Asp Glu Phe Ile Asp Gln Trp Leu Gln Glu Ala LysGln Lys Ile Lys Asp Glu Phe Ile Asp Gln Trp Leu Gln Glu Ala Lys

85 90 95 85 90 95

Pro Val Leu Asn Asp Gln Leu Lys Lys Leu Val Lys Asn Tyr Leu LeuPro Val Leu Asn Asp Gln Leu Lys Lys Leu Val Lys Asn Tyr Leu Leu

100 105 110 100 105 110

Lys Ser Gln Lys Glu Phe Ser Thr Lys Lys Met Val Arg Ile Met MetLys Ser Gln Lys Glu Phe Ser Thr Lys Lys Met Val Arg Ile Met Met

115 120 125 115 120 125

Ala Gly Ile Asp Lys Lys Glu Ile Asn Ile Asp Leu Asp Arg Asp LeuAla Gly Ile Asp Lys Lys Glu Ile Asn Ile Asp Leu Asp Arg Asp Leu

130 135 140 130 135 140

Val Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe AlaVal Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala

145 150 155 160145 150 155 160

Ser Val Gly Asn Gly Ile Ser Leu His Asn Met Lys Tyr Gln Thr ArgSer Val Gly Asn Gly Ile Ser Leu His Asn Met Lys Tyr Gln Thr Arg

165 170 175 165 170 175

Lys Arg Glu Thr Ile Phe Ala Gln Phe Ile Phe Lys Tyr Asn Lys AspLys Arg Glu Thr Ile Phe Ala Gln Phe Ile Phe Lys Tyr Asn Lys Asp

180 185 190 180 185 190

Tyr Lys Thr Thr Pro His Trp Phe Asp Arg Phe Asp His Gly Ser IleTyr Lys Thr Thr Pro His Trp Phe Asp Arg Phe Asp His Gly Ser Ile

195 200 205 195 200 205

Glu Gly Gly Asp Val Phe Val Tyr Thr Lys Asp Thr Leu Val Ile GlyGlu Gly Gly Asp Val Phe Val Tyr Thr Lys Asp Thr Leu Val Ile Gly

210 215 220 210 215 220

Ile Ser Glu Arg Thr Thr Lys Glu Ala Val Leu Asn Ile Ala Lys LysIle Ser Glu Arg Thr Thr Lys Glu Ala Val Leu Asn Ile Ala Lys Lys

225 230 235 240225 230 235 240

Ile Lys Ala Asn Thr Asp Ser Lys Phe Lys Lys Ile Val Ala Ile AsnIle Lys Ala Asn Thr Asp Ser Lys Phe Lys Lys Ile Val Ala Ile Asn

245 250 255 245 250 255

Val Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Ile Thr MetVal Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Ile Thr Met

260 265 270 260 265 270

Val Asp His Asp Lys Phe Leu Tyr Ser Pro Asn Met Met Lys Ser LeuVal Asp His Asp Lys Phe Leu Tyr Ser Pro Asn Met Met Lys Ser Leu

275 280 285 275 280 285

Lys Phe Trp Leu Ile Asp Leu Ser Lys Glu Ile Lys Met Val Glu LeuLys Phe Trp Leu Ile Asp Leu Ser Lys Glu Ile Lys Met Val Glu Leu

290 295 300 290 295 300

Glu Glu Ser Leu Ser Asn Met Leu Glu Ala Ile Ile Gly Lys Lys ProGlu Glu Ser Leu Ser Asn Met Leu Glu Ala Ile Ile Gly Lys Lys Pro

305 310 315 320305 310 315 320

Ile Leu Ile Pro Ile Ala Gly Lys Asn Ala Ser Gln Leu Asp Ile AspIle Leu Ile Pro Ile Ala Gly Lys Asn Ala Ser Gln Leu Asp Ile Asp

325 330 335 325 330 335

Ile Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro GlyIle Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly

340 345 350 340 345 350

Val Val Val Gly Tyr Ser Arg Asn Lys Leu Thr Gln Lys Ala Leu GluVal Val Val Gly Tyr Ser Arg Asn Lys Leu Thr Gln Lys Ala Leu Glu

355 360 365 355 360 365

Asp Ala Gly Val Lys Val Leu Ser Phe Asp Gly Asn Gln Leu Ser LeuAsp Ala Gly Val Lys Val Leu Ser Phe Asp Gly Asn Gln Leu Ser Leu

370 375 380 370 375 380

Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu AspGly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp

385 390 395 400385 390 395 400

Ile LysIle Lys

<210> 16<210> 16

<211> 438<211> 438

<212> PRT<212> PRT

<213> 肺炎支原体（Mycoplasma pneumoniae）<213> Mycoplasma pneumoniae

<400> 16<400> 16

Met Ser Lys Lys Gln Leu Val Lys Thr Asp Gly His Asn Gln Leu AspMet Ser Lys Lys Gln Leu Val Lys Thr Asp Gly His Asn Gln Leu Asp

1 5 10 151 5 10 15

Gln Pro Asn Thr Lys Ala Leu Gln Leu Lys Lys Lys Gln Phe Asn SerGln Pro Asn Thr Lys Ala Leu Gln Leu Lys Lys Lys Gln Phe Asn Ser

20 25 30 20 25 30

Gly Val Arg Val Thr Ser Glu Ile Ser Phe Leu Arg Glu Val Ile AlaGly Val Arg Val Thr Ser Glu Ile Ser Phe Leu Arg Glu Val Ile Ala

35 40 45 35 40 45

His His Pro Gly Ile Glu Thr Glu Arg Val Ile Asp Asn Gln Thr PheHis His Pro Gly Ile Glu Thr Glu Arg Val Ile Asp Asn Gln Thr Phe

50 55 60 50 55 60

Gly Ser Ala Met Tyr Leu Glu Arg Ala Gln Lys Glu His Gln Leu PheGly Ser Ala Met Tyr Leu Glu Arg Ala Gln Lys Glu His Gln Leu Phe

65 70 75 8065 70 75 80

Ile Lys Ile Leu Arg Gln His Gly Thr Lys Val His Tyr Leu Gln AspIle Lys Ile Leu Arg Gln His Gly Thr Lys Val His Tyr Leu Gln Asp

85 90 95 85 90 95

Leu Leu Leu Glu Ala Leu Ser Ala Ala Asp Pro Asn Val Arg Gln AspLeu Leu Leu Glu Ala Leu Ser Ala Ala Asp Pro Asn Val Arg Gln Asp

100 105 110 100 105 110

Phe Ile Lys Asn Phe Leu Leu Glu Ser Gly Ile Lys Ser Val Ser ThrPhe Ile Lys Asn Phe Leu Leu Glu Ser Gly Ile Lys Ser Val Ser Thr

115 120 125 115 120 125

Phe Glu Ala Cys Leu Asn Phe Phe Arg Ser Leu Asp Ser Leu Val AspPhe Glu Ala Cys Leu Asn Phe Phe Arg Ser Leu Asp Ser Leu Val Asp

130 135 140 130 135 140

Val Ile Lys Val Met Phe Gly Gly Ile Lys Val Ser Asp Val Pro ProVal Ile Lys Val Met Phe Gly Gly Ile Lys Val Ser Asp Val Pro Pro

145 150 155 160145 150 155 160

Ile Thr Pro Gln Arg Phe Ala Asp Ile His Val Ser Asn Ser Pro PheIle Thr Pro Gln Arg Phe Ala Asp Ile His Val Ser Asn Ser Pro Phe

165 170 175 165 170 175

Leu Ile Lys Pro Leu Ser Phe Ser Leu Tyr Pro His Lys Phe Phe AsnLeu Ile Lys Pro Leu Ser Phe Ser Leu Tyr Pro His Lys Phe Phe Asn

180 185 190 180 185 190

Thr Leu Gly Thr Gly Val Ala Leu Phe Val Thr Asn Asp Ser Glu LeuThr Leu Gly Thr Gly Val Ala Leu Phe Val Thr Asn Asp Ser Glu Leu

195 200 205 195 200 205

Lys Arg His Ser Leu Val Tyr Glu Tyr Ile Met Arg Phe His Pro ArgLys Arg His Ser Leu Val Tyr Glu Tyr Ile Met Arg Phe His Pro Arg

210 215 220 210 215 220

Phe Asp Gly Val Lys Leu Tyr Thr Asn Arg Asp Phe Lys Asn Cys LeuPhe Asp Gly Val Lys Leu Tyr Thr Asn Arg Asp Phe Lys Asn Cys Leu

225 230 235 240225 230 235 240

Ile Asn Ser Ser Asp Ile Ile Gln Ile Ser Asn Glu Ile Leu Leu IleIle Asn Ser Ser Asp Ile Ile Gln Ile Ser Asn Glu Ile Leu Leu Ile

245 250 255 245 250 255

Gly Ile Ser His Asp Thr Asp Val Leu Gly Ile Glu Ser Leu Ala ArgGly Ile Ser His Asp Thr Asp Val Leu Gly Ile Glu Ser Leu Ala Arg

260 265 270 260 265 270

Asn Leu Leu Ser Asp His Thr Asn Pro Ile Lys Gln Ile Ile Ala IleAsn Leu Leu Ser Asp His Thr Asn Pro Ile Lys Gln Ile Ile Ala Ile

275 280 285 275 280 285

Asn Ile His Lys Phe Gly Ala Lys Thr Asn Leu Asn Lys Leu Ile AlaAsn Ile His Lys Phe Gly Ala Lys Thr Asn Leu Asn Lys Leu Ile Ala

290 295 300 290 295 300

Met Val Asp Val Asp Lys Phe Ile Ile Ala Arg Lys Val Leu Gln AlaMet Val Asp Val Asp Lys Phe Ile Ile Ala Arg Lys Val Leu Gln Ala

305 310 315 320305 310 315 320

Thr Glu Ile Phe Glu Leu Thr Ala Thr Ala Gln Arg Asp Val Asp GlyThr Glu Ile Phe Glu Leu Thr Ala Thr Ala Gln Arg Asp Val Asp Gly

325 330 335 325 330 335

Leu Ala Gln Ile Lys Phe Lys Pro Leu Lys Phe Asn Phe Gly Glu IleLeu Ala Gln Ile Lys Phe Lys Pro Leu Lys Phe Asn Phe Gly Glu Ile

340 345 350 340 345 350

Ile Glu Ala Ile Ile Asp Lys Gln Pro Arg Phe Val Ile Ile Gly GlyIle Glu Ala Ile Ile Asp Lys Gln Pro Arg Phe Val Ile Ile Gly Gly

355 360 365 355 360 365

Gly Asp Glu Val Ala Glu Arg Lys Glu Leu Leu Asp Cys Gly Met GlyGly Asp Glu Val Ala Glu Arg Lys Glu Leu Leu Asp Cys Gly Met Gly

370 375 380 370 375 380

Val Leu Asn Leu Ser Pro Gly Glu Ile Val Val Phe Asp Arg Asn HisVal Leu Asn Leu Ser Pro Gly Glu Ile Val Val Phe Asp Arg Asn His

385 390 395 400385 390 395 400

Tyr Thr Asn Asn Leu Leu Asn Glu Leu Gly Leu Ile Ile His Lys IleTyr Thr Asn Asn Leu Leu Asn Glu Leu Gly Leu Ile Ile His Lys Ile

405 410 415 405 410 415

Pro Ala Ser Glu Leu Ser Arg Gly Pro Ser Gly Pro Leu Glu Met ValPro Ala Ser Glu Leu Ser Arg Gly Pro Ser Gly Pro Leu Glu Met Val

420 425 430 420 425 430

Cys Ser Leu Trp Arg GluCys Ser Leu Trp Arg Glu

435 435

<210> 17<210> 17

<211> 409<211> 409

<212> PRT<212> PRT

<213> 运动支原体（Mycoplasma mobile）<213> Mycoplasma mobile

<400> 17<400> 17

Met Lys Asp Thr Lys Asp Ile Ile Asn Val Phe Ser Glu Ile Gly GluMet Lys Asp Thr Lys Asp Ile Ile Asn Val Phe Ser Glu Ile Gly Glu

1 5 10 151 5 10 15

Leu Lys Lys Val Leu Ile His Thr Pro Gly Asn Glu Leu Lys Tyr ValLeu Lys Lys Val Leu Ile His Thr Pro Gly Asn Glu Leu Lys Tyr Val

20 25 30 20 25 30

Ser Pro Tyr Arg Leu Asp Glu Leu Leu Phe Ser Asn Val Leu Glu TrpSer Pro Tyr Arg Leu Asp Glu Leu Leu Phe Ser Asn Val Leu Glu Trp

35 40 45 35 40 45

Arg Glu Ala Lys Lys Glu His Asn Glu Phe Ile Gln Lys Leu Lys SerArg Glu Ala Lys Lys Glu His Asn Glu Phe Ile Gln Lys Leu Lys Ser

50 55 60 50 55 60

Glu Gly Val Glu Pro Val Glu Leu Thr Asp Leu Val Ala Glu Ser PheGlu Gly Val Glu Pro Val Glu Leu Thr Asp Leu Val Ala Glu Ser Phe

65 70 75 8065 70 75 80

Glu Glu Ser Ser Ile Lys Val Lys Asn Asp Phe Ile Arg Gln Tyr LeuGlu Glu Ser Ser Ile Lys Val Lys Asn Asp Phe Ile Arg Gln Tyr Leu

85 90 95 85 90 95

Asp Glu Ala Thr Pro Ile Leu Asp Gly Leu Thr Lys Gln Lys Leu LeuAsp Glu Ala Thr Pro Ile Leu Asp Gly Leu Thr Lys Gln Lys Leu Leu

100 105 110 100 105 110

Pro Phe Phe Leu Asp Ile Lys His Ser Thr Arg Lys Thr Ile Glu LeuPro Phe Phe Leu Asp Ile Lys His Ser Thr Arg Lys Thr Ile Glu Leu

115 120 125 115 120 125

Met Met Ser Gly Ile Thr Gln Lys Asp Ile Ser Ile Ser His Ile GluMet Met Ser Gly Ile Thr Gln Lys Asp Ile Ser Ile Ser His Ile Glu

130 135 140 130 135 140

Arg Glu Leu Ile Ile Asp Pro Met Pro Asn Leu Tyr Phe Ser Arg AspArg Glu Leu Ile Ile Asp Pro Met Pro Asn Leu Tyr Phe Ser Arg Asp

145 150 155 160145 150 155 160

Asn Phe Ile Ser Ile Gly Asn Ser Val Ile Ile Ser Asn Met Lys TyrAsn Phe Ile Ser Ile Gly Asn Ser Val Ile Ile Ser Asn Met Lys Tyr

165 170 175 165 170 175

Lys Thr Arg Lys Arg Glu Thr Ile Phe Thr Asp Phe Ile Phe Lys AsnLys Thr Arg Lys Arg Glu Thr Ile Phe Thr Asp Phe Ile Phe Lys Asn

180 185 190 180 185 190

His Pro Leu Tyr Lys Lys Val Asn Met Ala Phe Glu Arg Lys Asp LeuHis Pro Leu Tyr Lys Lys Val Asn Met Ala Phe Glu Arg Lys Asp Leu

195 200 205 195 200 205

Asn Asn Gln Ile Ser Ile Ile Glu Gly Gly Asp Val Leu Val Tyr SerAsn Asn Gln Ile Ser Ile Ile Glu Gly Gly Asp Val Leu Val Tyr Ser

210 215 220 210 215 220

Lys Glu Ile Leu Ile Ile Gly Ile Ser Glu Arg Thr Thr Met Ser AlaLys Glu Ile Leu Ile Ile Gly Ile Ser Glu Arg Thr Thr Met Ser Ala

225 230 235 240225 230 235 240

Ile Leu Glu Leu Ala Glu Asn Phe Lys Lys Thr Lys Arg Ser Phe LysIle Leu Glu Leu Ala Glu Asn Phe Lys Lys Thr Lys Arg Ser Phe Lys

245 250 255 245 250 255

Lys Ile Tyr Gly Val Glu Val Pro Lys Met Lys Asn Leu Met His LeuLys Ile Tyr Gly Val Glu Val Pro Lys Met Lys Asn Leu Met His Leu

260 265 270 260 265 270

Asp Thr Trp Leu Thr Met Ile Asp Tyr Asp Lys Phe Ile Tyr Ser ProAsp Thr Trp Leu Thr Met Ile Asp Tyr Asp Lys Phe Ile Tyr Ser Pro

275 280 285 275 280 285

Asn Val Leu Thr Asp Leu Lys Phe Trp Glu Ile Asn Leu Asp Tyr GluAsn Val Leu Thr Asp Leu Lys Phe Trp Glu Ile Asn Leu Asp Tyr Glu

290 295 300 290 295 300

Lys Ile Ser Ser Lys Glu Leu His Ala Ser Leu Ser Glu Phe Leu LysLys Ile Ser Ser Lys Glu Leu His Ala Ser Leu Ser Glu Phe Leu Lys

305 310 315 320305 310 315 320

Leu Ile Ile Gly Lys Asp Pro Ile Leu Ile Pro Ile Gly Gly Lys GlyLeu Ile Ile Gly Lys Asp Pro Ile Leu Ile Pro Ile Gly Gly Lys Gly

325 330 335 325 330 335

Ala Ser Gln Ile Thr Ile Asp Ile Glu Thr Asn Phe Val Ala Ala AsnAla Ser Gln Ile Thr Ile Asp Ile Glu Thr Asn Phe Val Ala Ala Asn

340 345 350 340 345 350

Tyr Leu Val Ile Arg Pro Gly Val Val Ile Gly Tyr Ser Arg Asn TyrTyr Leu Val Ile Arg Pro Gly Val Val Ile Gly Tyr Ser Arg Asn Tyr

355 360 365 355 360 365

Glu Thr Gln Lys Ala Leu Glu Gly His Gly Val Lys Val Ile Ala PheGlu Thr Gln Lys Ala Leu Glu Gly His Gly Val Lys Val Ile Ala Phe

370 375 380 370 375 380

Glu Gly Asn Gln Leu Ser Leu Gly Met Gly Ser Ser Arg Cys Met SerGlu Gly Asn Gln Leu Ser Leu Gly Met Gly Ser Ser Arg Cys Met Ser

385 390 395 400385 390 395 400

Met Pro Leu Ile Arg Ser Asn Leu LysMet Pro Leu Ile Arg Ser Asn Leu Lys

405 405

<210> 18<210> 18

<211> 411<211> 411

<212> PRT<212> PRT

<213> 化脓性链球菌（Streptococcus pyogenes）<213> Streptococcus pyogenes

<400> 18<400> 18

Met Thr Ala Gln Thr Pro Ile His Val Tyr Ser Glu Ile Gly Lys LeuMet Thr Ala Gln Thr Pro Ile His Val Tyr Ser Glu Ile Gly Lys Leu

1 5 10 151 5 10 15

Lys Lys Val Leu Leu His Arg Pro Gly Lys Glu Ile Glu Asn Leu MetLys Lys Val Leu Leu His Arg Pro Gly Lys Glu Ile Glu Asn Leu Met

20 25 30 20 25 30

Pro Asp Tyr Leu Glu Arg Leu Leu Phe Asp Asp Ile Pro Phe Leu GluPro Asp Tyr Leu Glu Arg Leu Leu Phe Asp Asp Ile Pro Phe Leu Glu

35 40 45 35 40 45

Asp Ala Gln Lys Glu His Asp Ala Phe Ala Gln Ala Leu Arg Asp GluAsp Ala Gln Lys Glu His Asp Ala Phe Ala Gln Ala Leu Arg Asp Glu

50 55 60 50 55 60

Gly Ile Glu Val Leu Tyr Leu Glu Thr Leu Ala Ala Glu Ser Leu ValGly Ile Glu Val Leu Tyr Leu Glu Thr Leu Ala Ala Glu Ser Leu Val

65 70 75 8065 70 75 80

Thr Pro Glu Ile Arg Glu Ala Phe Ile Asp Glu Tyr Leu Ser Glu AlaThr Pro Glu Ile Arg Glu Ala Phe Ile Asp Glu Tyr Leu Ser Glu Ala

85 90 95 85 90 95

Asn Ile Arg Gly Arg Ala Thr Lys Lys Ala Ile Arg Glu Leu Leu MetAsn Ile Arg Gly Arg Ala Thr Lys Lys Ala Ile Arg Glu Leu Leu Met

100 105 110 100 105 110

Ala Ile Glu Asp Asn Gln Glu Leu Ile Glu Lys Thr Met Ala Gly ValAla Ile Glu Asp Asn Gln Glu Leu Ile Glu Lys Thr Met Ala Gly Val

115 120 125 115 120 125

Gln Lys Ser Glu Leu Pro Glu Ile Pro Ala Ser Glu Lys Gly Leu ThrGln Lys Ser Glu Leu Pro Glu Ile Pro Ala Ser Glu Lys Gly Leu Thr

130 135 140 130 135 140

Asp Leu Val Glu Ser Asn Tyr Pro Phe Ala Ile Asp Pro Met Pro AsnAsp Leu Val Glu Ser Asn Tyr Pro Phe Ala Ile Asp Pro Met Pro Asn

145 150 155 160145 150 155 160

Leu Tyr Phe Thr Arg Asp Pro Phe Ala Thr Ile Gly Thr Gly Val SerLeu Tyr Phe Thr Arg Asp Pro Phe Ala Thr Ile Gly Thr Gly Val Ser

165 170 175 165 170 175

Leu Asn His Met Phe Ser Glu Thr Arg Asn Arg Glu Thr Leu Tyr GlyLeu Asn His Met Phe Ser Glu Thr Arg Asn Arg Glu Thr Leu Tyr Gly

180 185 190 180 185 190

Lys Tyr Ile Phe Thr His His Pro Ile Tyr Gly Gly Gly Lys Val ProLys Tyr Ile Phe Thr His His Pro Ile Tyr Gly Gly Gly Lys Val Pro

195 200 205 195 200 205

Met Val Tyr Asp Arg Asn Glu Thr Thr Arg Ile Glu Gly Gly Asp GluMet Val Tyr Asp Arg Asn Glu Thr Thr Arg Ile Glu Gly Gly Asp Glu

210 215 220 210 215 220

Leu Val Leu Ser Lys Asp Val Leu Ala Val Gly Ile Ser Gln Arg ThrLeu Val Leu Ser Lys Asp Val Leu Ala Val Gly Ile Ser Gln Arg Thr

225 230 235 240225 230 235 240

Asp Ala Ala Ser Ile Glu Lys Leu Leu Val Asn Ile Phe Lys Gln AsnAsp Ala Ala Ser Ile Glu Lys Leu Leu Val Asn Ile Phe Lys Gln Asn

245 250 255 245 250 255

Leu Gly Phe Lys Lys Val Leu Ala Phe Glu Phe Ala Asn Asn Arg LysLeu Gly Phe Lys Lys Val Leu Ala Phe Glu Phe Ala Asn Asn Arg Lys

260 265 270 260 265 270

Phe Met His Leu Asp Thr Val Phe Thr Met Val Asp Tyr Asp Lys PhePhe Met His Leu Asp Thr Val Phe Thr Met Val Asp Tyr Asp Lys Phe

275 280 285 275 280 285

Thr Ile His Pro Glu Ile Glu Gly Asp Leu Arg Val Tyr Ser Val ThrThr Ile His Pro Glu Ile Glu Gly Asp Leu Arg Val Tyr Ser Val Thr

290 295 300 290 295 300

Tyr Asp Asn Glu Glu Leu His Ile Val Glu Glu Lys Gly Asp Leu AlaTyr Asp Asn Glu Glu Leu His Ile Val Glu Glu Lys Gly Asp Leu Ala

305 310 315 320305 310 315 320

Glu Leu Leu Ala Ala Asn Leu Gly Val Glu Lys Val Asp Leu Ile ArgGlu Leu Leu Ala Ala Asn Leu Gly Val Glu Lys Val Asp Leu Ile Arg

325 330 335 325 330 335

Cys Gly Gly Asp Asn Leu Val Ala Ala Gly Arg Glu Gln Trp Asn AspCys Gly Gly Asp Asn Leu Val Ala Ala Gly Arg Glu Gln Trp Asn Asp

340 345 350 340 345 350

Gly Ser Asn Thr Leu Thr Ile Ala Pro Gly Val Val Val Val Tyr AsnGly Ser Asn Thr Leu Thr Ile Ala Pro Gly Val Val Val Val Tyr Asn

355 360 365 355 360 365

Arg Asn Thr Ile Thr Asn Ala Ile Leu Glu Ser Lys Gly Leu Lys LeuArg Asn Thr Ile Thr Asn Ala Ile Leu Glu Ser Lys Gly Leu Lys Leu

370 375 380 370 375 380

Ile Lys Ile His Gly Ser Glu Leu Val Arg Gly Arg Gly Gly Pro ArgIle Lys Ile His Gly Ser Glu Leu Val Arg Gly Arg Gly Gly Pro Arg

385 390 395 400385 390 395 400

Cys Met Ser Met Pro Phe Glu Arg Glu Asp IleCys Met Ser Met Pro Phe Glu Arg Glu Asp Ile

405 410 405 410

<210> 19<210> 19

<211> 408<211> 408

<212> PRT<212> PRT

<213> 粪肠球菌（Enterococcus faecalis）<213> Enterococcus faecalis

<400> 19<400> 19

Met Ser His Pro Ile Asn Val Phe Ser Glu Ile Gly Lys Leu Lys ThrMet Ser His Pro Ile Asn Val Phe Ser Glu Ile Gly Lys Leu Lys Thr

1 5 10 151 5 10 15

Val Met Leu His Arg Pro Gly Lys Glu Leu Glu Asn Leu Met Pro AspVal Met Leu His Arg Pro Gly Lys Glu Leu Glu Asn Leu Met Pro Asp

20 25 30 20 25 30

Tyr Leu Glu Arg Leu Leu Phe Asp Asp Ile Pro Phe Leu Glu Lys AlaTyr Leu Glu Arg Leu Leu Phe Asp Asp Ile Pro Phe Leu Glu Lys Ala

35 40 45 35 40 45

Gln Ala Glu His Asp Ala Phe Ala Glu Leu Leu Arg Ser Lys Asp IleGln Ala Glu His Asp Ala Phe Ala Glu Leu Leu Arg Ser Lys Asp Ile

50 55 60 50 55 60

Glu Val Val Tyr Leu Glu Asp Leu Ala Ala Glu Ala Leu Ile Asn GluGlu Val Val Tyr Leu Glu Asp Leu Ala Ala Glu Ala Leu Ile Asn Glu

65 70 75 8065 70 75 80

Glu Val Arg Arg Gln Phe Ile Asp Gln Phe Leu Glu Glu Ala Asn IleGlu Val Arg Arg Gln Phe Ile Asp Gln Phe Leu Glu Glu Ala Asn Ile

85 90 95 85 90 95

Arg Ser Glu Ser Ala Lys Glu Lys Val Arg Glu Leu Met Leu Glu IleArg Ser Glu Ser Ala Lys Glu Lys Val Arg Glu Leu Met Leu Glu Ile

100 105 110 100 105 110

Asp Asp Asn Glu Glu Leu Ile Gln Lys Ala Ile Ala Gly Ile Gln LysAsp Asp Asn Glu Glu Leu Ile Gln Lys Ala Ile Ala Gly Ile Gln Lys

115 120 125 115 120 125

Gln Glu Leu Pro Lys Tyr Glu Gln Glu Phe Leu Thr Asp Met Val GluGln Glu Leu Pro Lys Tyr Glu Gln Glu Phe Leu Thr Asp Met Val Glu

130 135 140 130 135 140

Ala Asp Tyr Pro Phe Ile Ile Asp Pro Met Pro Asn Leu Tyr Phe ThrAla Asp Tyr Pro Phe Ile Ile Asp Pro Met Pro Asn Leu Tyr Phe Thr

145 150 155 160145 150 155 160

Arg Asp Asn Phe Ala Thr Met Gly His Gly Ile Ser Leu Asn His MetArg Asp Asn Phe Ala Thr Met Gly His Gly Ile Ser Leu Asn His Met

165 170 175 165 170 175

Tyr Ser Val Thr Arg Gln Arg Glu Thr Ile Phe Gly Gln Tyr Ile PheTyr Ser Val Thr Arg Gln Arg Glu Thr Ile Phe Gly Gln Tyr Ile Phe

180 185 190 180 185 190

Asp Tyr His Pro Arg Phe Ala Gly Lys Glu Val Pro Arg Val Tyr AspAsp Tyr His Pro Arg Phe Ala Gly Lys Glu Val Pro Arg Val Tyr Asp

195 200 205 195 200 205

Arg Ser Glu Ser Thr Arg Ile Glu Gly Gly Asp Glu Leu Ile Leu SerArg Ser Glu Ser Thr Arg Ile Glu Gly Gly Asp Glu Leu Ile Leu Ser

210 215 220 210 215 220

Lys Glu Val Val Ala Ile Gly Ile Ser Gln Arg Thr Asp Ala Ala SerLys Glu Val Val Ala Ile Gly Ile Ser Gln Arg Thr Asp Ala Ala Ser

225 230 235 240225 230 235 240

Ile Glu Lys Ile Ala Arg Asn Ile Phe Glu Gln Lys Leu Gly Phe LysIle Glu Lys Ile Ala Arg Asn Ile Phe Glu Gln Lys Leu Gly Phe Lys

245 250 255 245 250 255

Asn Ile Leu Ala Phe Asp Ile Gly Glu His Arg Lys Phe Met His LeuAsn Ile Leu Ala Phe Asp Ile Gly Glu His Arg Lys Phe Met His Leu

260 265 270 260 265 270

Asp Thr Val Phe Thr Met Ile Asp Tyr Asp Lys Phe Thr Ile His ProAsp Thr Val Phe Thr Met Ile Asp Tyr Asp Lys Phe Thr Ile His Pro

275 280 285 275 280 285

Glu Ile Glu Gly Gly Leu Val Val Tyr Ser Ile Thr Glu Lys Ala AspGlu Ile Glu Gly Gly Leu Val Val Tyr Ser Ile Thr Glu Lys Ala Asp

290 295 300 290 295 300

Gly Asp Ile Gln Ile Thr Lys Glu Lys Asp Thr Leu Asp Asn Ile LeuGly Asp Ile Gln Ile Thr Lys Glu Lys Asp Thr Leu Asp Asn Ile Leu

305 310 315 320305 310 315 320

Cys Lys Tyr Leu His Leu Asp Asn Val Gln Leu Ile Arg Cys Gly AlaCys Lys Tyr Leu His Leu Asp Asn Val Gln Leu Ile Arg Cys Gly Ala

325 330 335 325 330 335

Gly Asn Leu Thr Ala Ala Ala Arg Glu Gln Trp Asn Asp Gly Ser AsnGly Asn Leu Thr Ala Ala Ala Arg Glu Gln Trp Asn Asp Gly Ser Asn

340 345 350 340 345 350

Thr Leu Ala Ile Ala Pro Gly Glu Val Val Val Tyr Asp Arg Asn ThrThr Leu Ala Ile Ala Pro Gly Glu Val Val Val Tyr Asp Arg Asn Thr

355 360 365 355 360 365

Ile Thr Asn Lys Ala Leu Glu Glu Ala Gly Val Lys Leu Asn Tyr IleIle Thr Asn Lys Ala Leu Glu Glu Ala Gly Val Lys Leu Asn Tyr Ile

370 375 380 370 375 380

Pro Gly Ser Glu Leu Val Arg Gly Arg Gly Gly Pro Arg Cys Met SerPro Gly Ser Glu Leu Val Arg Gly Arg Gly Gly Pro Arg Cys Met Ser

385 390 395 400385 390 395 400

Met Pro Leu Tyr Arg Glu Asp LeuMet Pro Leu Tyr Arg Glu Asp Leu

405 405

<210> 20<210> 20

<211> 403<211> 403

<212> PRT<212> PRT

<213> 公山羊支原体（Mycoplasma capricolum）<213> Mycoplasma capricolum

<400> 20<400> 20

Met Glu Lys Lys Ile Asn Val Phe Ser Glu Ile Gly Thr Leu Lys ThrMet Glu Lys Lys Ile Asn Val Phe Ser Glu Ile Gly Thr Leu Lys Thr

1 5 10 151 5 10 15

Val Leu Val His Arg Pro Gly Asp Glu Ile Glu Asn Leu Thr Pro GluVal Leu Val His Arg Pro Gly Asp Glu Ile Glu Asn Leu Thr Pro Glu

20 25 30 20 25 30

Leu Leu Glu Arg Leu Leu Phe Asp Asp Val Pro Phe Lys Asp Val AlaLeu Leu Glu Arg Leu Leu Phe Asp Asp Val Pro Phe Lys Asp Val Ala

35 40 45 35 40 45

Val Lys Glu His Asp Ala Phe Thr Lys Ile Met Arg Asp Asn Gly ValVal Lys Glu His Asp Ala Phe Thr Lys Ile Met Arg Asp Asn Gly Val

50 55 60 50 55 60

Glu Val Leu Tyr Ile Glu Lys Leu Ala Ala Glu Thr Leu Asp Gln HisGlu Val Leu Tyr Ile Glu Lys Leu Ala Ala Glu Thr Leu Asp Gln His

65 70 75 8065 70 75 80

Pro Asp Leu Arg Glu Lys Phe Ile Asp Gln Phe Ile Ser Glu Ala AsnPro Asp Leu Arg Glu Lys Phe Ile Asp Gln Phe Ile Ser Glu Ala Asn

85 90 95 85 90 95

Ile Glu Asp Lys Tyr Lys Glu Lys Tyr Arg Asp Phe Ile Ser Ser LeuIle Glu Asp Lys Tyr Lys Glu Lys Tyr Arg Asp Phe Ile Ser Ser Leu

100 105 110 100 105 110

Asp Asn Tyr Arg Met Ile Lys Lys Met Ile Ala Gly Thr Lys Lys LeuAsp Asn Tyr Arg Met Ile Lys Lys Met Ile Ala Gly Thr Lys Lys Leu

115 120 125 115 120 125

Glu Leu Gly Ile Asp Glu Gly Tyr Lys Ala Tyr Pro Phe Ile Ala AspGlu Leu Gly Ile Asp Glu Gly Tyr Lys Ala Tyr Pro Phe Ile Ala Asp

130 135 140 130 135 140

Pro Leu Pro Asn Val Leu Phe Gln Arg Asp Pro Phe Ser Ser Val GlyPro Leu Pro Asn Val Leu Phe Gln Arg Asp Pro Phe Ser Ser Val Gly

145 150 155 160145 150 155 160

Phe Gly Ile Thr Met Asn Arg Met Trp Ser Val Thr Arg Asn Arg GluPhe Gly Ile Thr Met Asn Arg Met Trp Ser Val Thr Arg Asn Arg Glu

165 170 175 165 170 175

Thr Ile Phe Pro Asp Leu Val Phe Lys His His Asn Arg Phe Ala AsnThr Ile Phe Pro Asp Leu Val Phe Lys His His Asn Arg Phe Ala Asn

180 185 190 180 185 190

Gln Val Pro Tyr Tyr Tyr Glu Arg Asp Trp Lys Glu Glu Thr Ile GluGln Val Pro Tyr Tyr Tyr Glu Arg Asp Trp Lys Glu Glu Thr Ile Glu

195 200 205 195 200 205

Gly Gly Asp Ile Leu Val Leu Asn Lys Glu Thr Leu Ile Ile Gly ValGly Gly Asp Ile Leu Val Leu Asn Lys Glu Thr Leu Ile Ile Gly Val

210 215 220 210 215 220

Thr Gln Arg Thr Thr Leu Lys Ala Ile Glu Lys Phe Ser Glu Arg LeuThr Gln Arg Thr Thr Leu Lys Ala Ile Glu Lys Phe Ser Glu Arg Leu

225 230 235 240225 230 235 240

Phe Asn Asp Pro Glu Ser Ser Tyr Ser Lys Val Ile Ala Leu Asp LeuPhe Asn Asp Pro Glu Ser Ser Tyr Ser Lys Val Ile Ala Leu Asp Leu

245 250 255 245 250 255

Pro Lys Ser Arg Ala Phe Met His Leu Asp Thr Val Phe Thr Asn IlePro Lys Ser Arg Ala Phe Met His Leu Asp Thr Val Phe Thr Asn Ile

260 265 270 260 265 270

Asp Tyr Asp Lys Phe Ile Ala His Pro Leu Ile Phe Asp Cys Ile AspAsp Tyr Asp Lys Phe Ile Ala His Pro Leu Ile Phe Asp Cys Ile Asp

275 280 285 275 280 285

Glu Phe Lys Ile Tyr Glu Val Ser Lys Gln Gly Thr Lys Glu Val LysGlu Phe Lys Ile Tyr Glu Val Ser Lys Gln Gly Thr Lys Glu Val Lys

290 295 300 290 295 300

Lys Thr Leu Ile Glu Leu Leu Ser Asp Ala Ala Gly Arg Glu Val GlnLys Thr Leu Ile Glu Leu Leu Ser Asp Ala Ala Gly Arg Glu Val Gln

305 310 315 320305 310 315 320

Ile Ile Arg Cys Gly Gly Asn Asp Val Val Gly Ala Ser Arg Glu GlnIle Ile Arg Cys Gly Gly Asn Asp Val Val Gly Ala Ser Arg Glu Gln

325 330 335 325 330 335

Trp Asn Asp Gly Thr Asn Val Val Ala Leu Arg Pro Gly Lys Val IleTrp Asn Asp Gly Thr Asn Val Val Ala Leu Arg Pro Gly Lys Val Ile

340 345 350 340 345 350

Ala Tyr Glu Arg Asn Trp Ile Thr Ile Asp Leu Leu Arg Lys Ala GlyAla Tyr Glu Arg Asn Trp Ile Thr Ile Asp Leu Leu Arg Lys Ala Gly

355 360 365 355 360 365

Val Glu Val Leu Thr Ile Ala Ser Ser Glu Leu Ser Arg Gly Arg GlyVal Glu Val Leu Thr Ile Ala Ser Ser Glu Leu Ser Arg Gly Arg Gly

370 375 380 370 375 380

Gly Pro Arg Cys Met Thr Met Pro Leu Trp Arg Glu Asp Leu Gln GluGly Pro Arg Cys Met Thr Met Pro Leu Trp Arg Glu Asp Leu Gln Glu

385 390 395 400385 390 395 400

Ile Lys ArgIle Lys Arg

<210> 21<210> 21

<211> 410<211> 410

<212> PRT<212> PRT

<213> 奥氏嗜热盐丝菌（Halothermothrix orenii）<213> Halothermothrix orenii

<400> 21<400> 21

Met Phe Lys Lys Ser Pro Leu Asn Val Thr Ser Glu Ile Gly Lys LeuMet Phe Lys Lys Ser Pro Leu Asn Val Thr Ser Glu Ile Gly Lys Leu

1 5 10 151 5 10 15

Lys Lys Val Leu Leu His Arg Pro Gly His Glu Ile Glu Asn Leu ThrLys Lys Val Leu Leu His Arg Pro Gly His Glu Ile Glu Asn Leu Thr

20 25 30 20 25 30

Pro Asp Leu Leu Glu Arg Leu Leu Phe Asp Asp Ile Pro Tyr Leu LysPro Asp Leu Leu Glu Arg Leu Leu Phe Asp Asp Ile Pro Tyr Leu Lys

35 40 45 35 40 45

Val Ala Gln Glu Glu His Asp Ala Phe Ala Gln Thr Leu Arg Asp AsnVal Ala Gln Glu Glu His Asp Ala Phe Ala Gln Thr Leu Arg Asp Asn

50 55 60 50 55 60

Gly Val Glu Val Leu Tyr Leu His Glu Leu Ala Ala Glu Ala Ile GlnGly Val Glu Val Leu Tyr Leu His Glu Leu Ala Ala Glu Ala Ile Gln

65 70 75 8065 70 75 80

Glu Asp Glu Ile Arg Lys Lys Phe Ile Glu Gln Phe Leu Asp Glu AlaGlu Asp Glu Ile Arg Lys Lys Phe Ile Glu Gln Phe Leu Asp Glu Ala

85 90 95 85 90 95

Gly Val Ile Gly Lys Gly Ala Arg Gln Val Leu Lys Glu Tyr Phe AlaGly Val Ile Gly Lys Gly Ala Arg Gln Val Leu Lys Glu Tyr Phe Ala

100 105 110 100 105 110

Asp Met Asp Asn Glu Thr Leu Ile Arg Lys Met Met Ala Gly Val ArgAsp Met Asp Asn Glu Thr Leu Ile Arg Lys Met Met Met Ala Gly Val Arg

115 120 125 115 120 125

Lys Lys Glu Ile Pro Ala Ile Glu Lys Val Ala Ser Leu Asn Asp MetLys Lys Glu Ile Pro Ala Ile Glu Lys Val Ala Ser Leu Asn Asp Met

130 135 140 130 135 140

Val Glu Glu Asp Tyr Pro Phe Val Leu Asp Pro Met Pro Asn Leu TyrVal Glu Glu Asp Tyr Pro Phe Val Leu Asp Pro Met Pro Asn Leu Tyr

145 150 155 160145 150 155 160

Phe Thr Arg Asp Pro Phe Ala Thr Ile Gly Thr Gly Ile Thr Leu AsnPhe Thr Arg Asp Pro Phe Ala Thr Ile Gly Thr Gly Ile Thr Leu Asn

165 170 175 165 170 175

His Met Arg Thr Glu Thr Arg Asn Arg Glu Val Ile Phe Ala Glu TyrHis Met Arg Thr Glu Thr Arg Asn Arg Glu Val Ile Phe Ala Glu Tyr

180 185 190 180 185 190

Ile Phe Ser Tyr His Pro Asp Phe Lys Asp Thr Glu Ile Pro Phe TrpIle Phe Ser Tyr His Pro Asp Phe Lys Asp Thr Glu Ile Pro Phe Trp

195 200 205 195 200 205

Phe Asp Arg Asn Glu Thr Thr Ser Ile Glu Gly Gly Asp Glu Leu IlePhe Asp Arg Asn Glu Thr Thr Ser Ile Glu Gly Gly Asp Glu Leu Ile

210 215 220 210 215 220

Leu Ser Asp Lys Val Leu Ala Met Gly Ile Ser Glu Arg Thr Asp AlaLeu Ser Asp Lys Val Leu Ala Met Gly Ile Ser Glu Arg Thr Asp Ala

225 230 235 240225 230 235 240

Ala Ser Ile Glu Lys Val Ala Arg Asn Ile Phe Thr Asp Gly Gln ProAla Ser Ile Glu Lys Val Ala Arg Asn Ile Phe Thr Asp Gly Gln Pro

245 250 255 245 250 255

Phe Glu Thr Ile Leu Ala Phe Lys Ile Pro Glu Lys Arg Ala Phe MetPhe Glu Thr Ile Leu Ala Phe Lys Ile Pro Glu Lys Arg Ala Phe Met

260 265 270 260 265 270

His Leu Asp Thr Val Phe Thr Met Val Asp Tyr Asp Lys Phe Thr IleHis Leu Asp Thr Val Phe Thr Met Val Asp Tyr Asp Lys Phe Thr Ile

275 280 285 275 280 285

His Ala Glu Ile Glu Gly Pro Leu Lys Val Tyr Ser Ile Thr Lys GlyHis Ala Glu Ile Glu Gly Pro Leu Lys Val Tyr Ser Ile Thr Lys Gly

290 295 300 290 295 300

Asp Asn Asp Glu Leu Lys Ile Asp Glu Glu Lys Ala Thr Leu Glu AspAsp Asn Asp Glu Leu Lys Ile Asp Glu Glu Lys Ala Thr Leu Glu Asp

305 310 315 320305 310 315 320

Thr Leu Lys Lys Tyr Leu Gly Leu Asp Glu Val Thr Leu Ile Arg CysThr Leu Lys Lys Tyr Leu Gly Leu Asp Glu Val Thr Leu Ile Arg Cys

325 330 335 325 330 335

Ala Gly Gly Asp Tyr Ile Asp Ala Gly Arg Glu Gln Trp Asn Asp GlyAla Gly Gly Asp Tyr Ile Asp Ala Gly Arg Glu Gln Trp Asn Asp Gly

340 345 350 340 345 350

Ser Asn Thr Leu Ala Ile Ala Pro Gly Glu Val Val Val Tyr Asn ArgSer Asn Thr Leu Ala Ile Ala Pro Gly Glu Val Val Val Tyr Asn Arg

355 360 365 355 360 365

Asn His Thr Thr Asn Arg Leu Leu Glu Glu His Gly Ile Lys Leu HisAsn His Thr Thr Asn Arg Leu Leu Glu Glu His Gly Ile Lys Leu His

370 375 380 370 375 380

Val Ile Pro Ser Ser Glu Leu Ser Arg Gly Arg Gly Gly Pro Arg CysVal Ile Pro Ser Ser Glu Leu Ser Arg Gly Arg Gly Gly Pro Arg Cys

385 390 395 400385 390 395 400

Met Ser Met Pro Leu Val Arg Glu Asp IleMet Ser Met Pro Leu Val Arg Glu Asp Ile

405 410 405 410

<210> 22<210> 22

<211> 411<211> 411

<212> PRT<212> PRT

<213> 金黄色葡萄球菌（Staphylococcus aureus）<213> Staphylococcus aureus

<400> 22<400> 22

Met Thr Asp Gly Pro Ile Lys Val Asn Ser Glu Ile Gly Ala Leu LysMet Thr Asp Gly Pro Ile Lys Val Asn Ser Glu Ile Gly Ala Leu Lys

1 5 10 151 5 10 15

Thr Val Leu Leu Lys Arg Pro Gly Lys Glu Leu Glu Asn Leu Val ProThr Val Leu Leu Lys Arg Pro Gly Lys Glu Leu Glu Asn Leu Val Pro

20 25 30 20 25 30

Asp Tyr Leu Asp Gly Leu Leu Phe Asp Asp Ile Pro Tyr Leu Glu ValAsp Tyr Leu Asp Gly Leu Leu Phe Asp Asp Ile Pro Tyr Leu Glu Val

35 40 45 35 40 45

Ala Gln Lys Glu His Asp His Phe Ala Gln Val Leu Arg Glu Glu GlyAla Gln Lys Glu His Asp His Phe Ala Gln Val Leu Arg Glu Glu Gly

50 55 60 50 55 60

Val Glu Val Leu Tyr Leu Glu Lys Leu Ala Ala Glu Ser Ile Glu AsnVal Glu Val Leu Tyr Leu Glu Lys Leu Ala Ala Glu Ser Ile Glu Asn

65 70 75 8065 70 75 80

Pro Gln Val Arg Ser Glu Phe Ile Asp Asp Val Leu Ala Glu Ser LysPro Gln Val Arg Ser Glu Phe Ile Asp Asp Val Leu Ala Glu Ser Lys

85 90 95 85 90 95

Lys Thr Ile Leu Gly His Glu Glu Glu Ile Lys Ala Leu Phe Ala ThrLys Thr Ile Leu Gly His Glu Glu Glu Glu Ile Lys Ala Leu Phe Ala Thr

100 105 110 100 105 110

Leu Ser Asn Gln Glu Leu Val Asp Lys Ile Met Ser Gly Val Arg LysLeu Ser Asn Gln Glu Leu Val Asp Lys Ile Met Ser Gly Val Arg Lys

115 120 125 115 120 125

Glu Glu Ile Asn Pro Lys Cys Thr His Leu Val Glu Tyr Met Asp AspGlu Glu Ile Asn Pro Lys Cys Thr His Leu Val Glu Tyr Met Asp Asp

130 135 140 130 135 140

Lys Tyr Pro Phe Tyr Leu Asp Pro Met Pro Asn Leu Tyr Phe Thr ArgLys Tyr Pro Phe Tyr Leu Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg

145 150 155 160145 150 155 160

Asp Pro Gln Ala Ser Ile Gly His Gly Ile Thr Ile Asn Arg Met PheAsp Pro Gln Ala Ser Ile Gly His Gly Ile Thr Ile Asn Arg Met Phe

165 170 175 165 170 175

Trp Arg Ala Arg Arg Arg Glu Ser Ile Phe Ile Gln Tyr Ile Val LysTrp Arg Ala Arg Arg Arg Glu Ser Ile Phe Ile Gln Tyr Ile Val Lys

180 185 190 180 185 190

His His Pro Arg Phe Lys Asp Ala Asn Ile Pro Ile Trp Leu Asp ArgHis His Pro Arg Phe Lys Asp Ala Asn Ile Pro Ile Trp Leu Asp Arg

195 200 205 195 200 205

Asp Cys Pro Phe Asn Ile Glu Gly Gly Asp Glu Leu Val Leu Ser LysAsp Cys Pro Phe Asn Ile Glu Gly Gly Asp Glu Leu Val Leu Ser Lys

210 215 220 210 215 220

Asp Val Leu Ala Ile Gly Val Ser Glu Arg Thr Ser Ala Gln Ala IleAsp Val Leu Ala Ile Gly Val Ser Glu Arg Thr Ser Ala Gln Ala Ile

225 230 235 240225 230 235 240

Glu Lys Leu Ala Arg Arg Ile Phe Glu Asn Pro Gln Ala Thr Phe LysGlu Lys Leu Ala Arg Arg Ile Phe Glu Asn Pro Gln Ala Thr Phe Lys

245 250 255 245 250 255

Lys Val Val Ala Ile Glu Ile Pro Thr Ser Arg Thr Phe Met His LeuLys Val Val Ala Ile Glu Ile Pro Thr Ser Arg Thr Phe Met His Leu

260 265 270 260 265 270

Asp Thr Val Phe Thr Met Ile Asp Tyr Asp Lys Phe Thr Met His SerAsp Thr Val Phe Thr Met Ile Asp Tyr Asp Lys Phe Thr Met His Ser

275 280 285 275 280 285

Ala Ile Leu Lys Ala Glu Gly Asn Met Asn Ile Phe Ile Ile Glu TyrAla Ile Leu Lys Ala Glu Gly Asn Met Asn Ile Phe Ile Ile Glu Tyr

290 295 300 290 295 300

Asp Asp Val Asn Lys Asp Ile Ala Ile Lys Gln Ser Ser His Leu LysAsp Asp Val Asn Lys Asp Ile Ala Ile Lys Gln Ser Ser His Leu Lys

305 310 315 320305 310 315 320

Asp Thr Leu Glu Asp Val Leu Gly Ile Asp Asp Ile Gln Phe Ile ProAsp Thr Leu Glu Asp Val Leu Gly Ile Asp Asp Ile Gln Phe Ile Pro

325 330 335 325 330 335

Thr Gly Asn Gly Asp Val Ile Asp Gly Ala Arg Glu Gln Trp Asn AspThr Gly Asn Gly Asp Val Ile Asp Gly Ala Arg Glu Gln Trp Asn Asp

340 345 350 340 345 350

Gly Ser Asn Thr Leu Cys Ile Arg Pro Gly Val Val Val Thr Tyr AspGly Ser Asn Thr Leu Cys Ile Arg Pro Gly Val Val Val Thr Tyr Asp

355 360 365 355 360 365

Arg Asn Tyr Val Ser Asn Asp Leu Leu Arg Gln Lys Gly Ile Lys ValArg Asn Tyr Val Ser Asn Asp Leu Leu Arg Gln Lys Gly Ile Lys Val

370 375 380 370 375 380

Ile Glu Ile Ser Gly Ser Glu Leu Val Arg Gly Arg Gly Gly Pro ArgIle Glu Ile Ser Gly Ser Glu Leu Val Arg Gly Arg Gly Gly Pro Arg

385 390 395 400385 390 395 400

Cys Met Ser Gln Pro Leu Phe Arg Glu Asp IleCys Met Ser Gln Pro Leu Phe Arg Glu Asp Ile

405 410 405 410

<210> 23<210> 23

<211> 417<211> 417

<212> PRT<212> PRT

<213> 杀香鱼假单胞菌（Pseudomonas plecoglossicida）<213> Pseudomonas plecoglossicida

<400> 23<400> 23

Met Ser Ala Glu Lys Gln Lys Tyr Gly Val His Ser Glu Ala Gly LysMet Ser Ala Glu Lys Gln Lys Tyr Gly Val His Ser Glu Ala Gly Lys

1 5 10 151 5 10 15

Leu Arg Lys Val Met Val Cys Ala Pro Gly Leu Ala His Lys Arg LeuLeu Arg Lys Val Met Val Cys Ala Pro Gly Leu Ala His Lys Arg Leu

20 25 30 20 25 30

Thr Pro Ser Asn Cys Asp Glu Leu Leu Phe Asp Asp Val Ile Trp ValThr Pro Ser Asn Cys Asp Glu Leu Leu Phe Asp Asp Val Ile Trp Val

35 40 45 35 40 45

Asp Gln Ala Lys Arg Asp His Phe Asp Phe Val Thr Lys Met Arg GluAsp Gln Ala Lys Arg Asp His Phe Asp Phe Val Thr Lys Met Arg Glu

50 55 60 50 55 60

Arg Gly Val Asp Val Leu Glu Met His Asn Leu Leu Thr Asp Ile ValArg Gly Val Asp Val Leu Glu Met His Asn Leu Leu Thr Asp Ile Val

65 70 75 8065 70 75 80

Gln Asn Pro Glu Ala Leu Lys Trp Ile Leu Asp Arg Lys Ile Thr ProGln Asn Pro Glu Ala Leu Lys Trp Ile Leu Asp Arg Lys Ile Thr Pro

85 90 95 85 90 95

Asp Thr Val Gly Val Gly Leu Thr Asn Glu Val Arg Ser Trp Leu GluAsp Thr Val Gly Val Gly Leu Thr Asn Glu Val Arg Ser Trp Leu Glu

100 105 110 100 105 110

Gly Gln Glu Pro Arg His Leu Ala Glu Phe Leu Ile Gly Gly Val AlaGly Gln Glu Pro Arg His Leu Ala Glu Phe Leu Ile Gly Gly Val Ala

115 120 125 115 120 125

Gly Gln Asp Leu Pro Glu Ser Glu Gly Ala Ser Val Val Lys Met TyrGly Gln Asp Leu Pro Glu Ser Glu Gly Ala Ser Val Val Lys Met Tyr

130 135 140 130 135 140

Asn Asp Tyr Leu Gly His Ser Ser Phe Ile Leu Pro Pro Leu Pro AsnAsn Asp Tyr Leu Gly His Ser Ser Phe Ile Leu Pro Pro Leu Pro Asn

145 150 155 160145 150 155 160

Thr Gln Phe Thr Arg Asp Thr Thr Cys Trp Ile Tyr Gly Gly Val ThrThr Gln Phe Thr Arg Asp Thr Thr Cys Trp Ile Tyr Gly Gly Val Thr

165 170 175 165 170 175

Leu Asn Pro Met Tyr Trp Pro Ala Arg Arg Gln Glu Thr Leu Leu ThrLeu Asn Pro Met Tyr Trp Pro Ala Arg Arg Gln Glu Thr Leu Leu Thr

180 185 190 180 185 190

Thr Ala Ile Tyr Lys Phe His Pro Glu Phe Thr Lys Ala Asp Phe GlnThr Ala Ile Tyr Lys Phe His Pro Glu Phe Thr Lys Ala Asp Phe Gln

195 200 205 195 200 205

Val Trp Tyr Gly Asp Pro Asp Gln Glu His Gly Gln Ala Thr Leu GluVal Trp Tyr Gly Asp Pro Asp Gln Glu His Gly Gln Ala Thr Leu Glu

210 215 220 210 215 220

Gly Gly Asp Val Met Pro Ile Gly Lys Gly Ile Val Leu Ile Gly MetGly Gly Asp Val Met Pro Ile Gly Lys Gly Ile Val Leu Ile Gly Met

225 230 235 240225 230 235 240

Gly Glu Arg Thr Ser Arg Gln Ala Ile Gly Gln Leu Ala Gln Asn LeuGly Glu Arg Thr Ser Arg Gln Ala Ile Gly Gln Leu Ala Gln Asn Leu

245 250 255 245 250 255

Phe Ala Lys Gly Ala Val Glu Gln Val Ile Val Ala Gly Leu Pro LysPhe Ala Lys Gly Ala Val Glu Gln Val Ile Val Ala Gly Leu Pro Lys

260 265 270 260 265 270

Ser Arg Ala Ala Met His Leu Asp Thr Val Phe Ser Phe Cys Asp ArgSer Arg Ala Ala Met His Leu Asp Thr Val Phe Ser Phe Cys Asp Arg

275 280 285 275 280 285

Asp Leu Val Thr Val Phe Pro Glu Val Val Arg Glu Ile Val Pro PheAsp Leu Val Thr Val Phe Pro Glu Val Val Arg Glu Ile Val Pro Phe

290 295 300 290 295 300

Ile Ile Arg Pro Asp Glu Ser Lys Pro Tyr Gly Met Asp Val Arg ArgIle Ile Arg Pro Asp Glu Ser Lys Pro Tyr Gly Met Asp Val Arg Arg

305 310 315 320305 310 315 320

Glu Asn Lys Ser Phe Ile Glu Val Val Gly Glu Gln Leu Gly Val LysGlu Asn Lys Ser Phe Ile Glu Val Val Gly Glu Gln Leu Gly Val Lys

325 330 335 325 330 335

Leu Arg Val Val Glu Thr Gly Gly Asn Ser Phe Ala Ala Glu Arg GluLeu Arg Val Val Glu Thr Gly Gly Asn Ser Phe Ala Ala Glu Arg Glu

340 345 350 340 345 350

Gln Trp Asp Asp Gly Asn Asn Val Val Ala Leu Glu Pro Gly Val ValGln Trp Asp Asp Gly Asn Asn Val Val Ala Leu Glu Pro Gly Val Val

355 360 365 355 360 365

Ile Gly Tyr Asp Arg Asn Thr Tyr Thr Asn Thr Leu Leu Arg Lys AlaIle Gly Tyr Asp Arg Asn Thr Tyr Thr Asn Thr Leu Leu Arg Lys Ala

370 375 380 370 375 380

Gly Ile Glu Val Ile Thr Ile Ser Ala Gly Glu Leu Gly Arg Gly ArgGly Ile Glu Val Ile Thr Ile Ser Ala Gly Glu Leu Gly Arg Gly Arg

385 390 395 400385 390 395 400

Gly Gly Gly His Cys Met Thr Cys Pro Ile Val Arg Asp Pro Ile AsnGly Gly Gly His Cys Met Thr Cys Pro Ile Val Arg Asp Pro Ile Asn

405 410 415 405 410 415

TyrTyr

<210> 24<210> 24

<211> 417<211> 417

<212> PRT<212> PRT

<213> 恶臭假单胞菌（Pseudomonas putida）<213> Pseudomonas putida

<400> 24<400> 24

Met Ser Ala Glu Lys Gln Lys Tyr Gly Val His Ser Glu Ala Gly LysMet Ser Ala Glu Lys Gln Lys Tyr Gly Val His Ser Glu Ala Gly Lys

1 5 10 151 5 10 15

Leu Arg Lys Val Met Val Cys Ala Pro Gly Leu Ala His Lys Arg LeuLeu Arg Lys Val Met Val Cys Ala Pro Gly Leu Ala His Lys Arg Leu

20 25 30 20 25 30

Thr Pro Ser Asn Cys Asp Glu Leu Leu Phe Asp Asp Val Ile Trp ValThr Pro Ser Asn Cys Asp Glu Leu Leu Phe Asp Asp Val Ile Trp Val

35 40 45 35 40 45

Asp Gln Ala Lys Arg Asp His Phe Asp Phe Val Thr Lys Met Arg GluAsp Gln Ala Lys Arg Asp His Phe Asp Phe Val Thr Lys Met Arg Glu

50 55 60 50 55 60

Arg Gly Val Asp Val Leu Glu Met His Asn Leu Leu Thr Asp Ile ValArg Gly Val Asp Val Leu Glu Met His Asn Leu Leu Thr Asp Ile Val

65 70 75 8065 70 75 80

Gln Asn Lys Asp Ala Leu Lys Trp Ile Leu Asp Arg Lys Ile Thr ProGln Asn Lys Asp Ala Leu Lys Trp Ile Leu Asp Arg Lys Ile Thr Pro

85 90 95 85 90 95

Asp Thr Val Gly Val Gly Leu Thr Asn Glu Val Arg Ser Trp Leu GluAsp Thr Val Gly Val Gly Leu Thr Asn Glu Val Arg Ser Trp Leu Glu

100 105 110 100 105 110

Gly Leu Glu Pro Arg His Leu Ala Glu Phe Leu Ile Gly Gly Val AlaGly Leu Glu Pro Arg His Leu Ala Glu Phe Leu Ile Gly Gly Val Ala

115 120 125 115 120 125

Gly Gln Asp Leu Pro Gln Ser Glu Gly Ala Asp Val Val Lys Met TyrGly Gln Asp Leu Pro Gln Ser Glu Gly Ala Asp Val Val Lys Met Tyr

130 135 140 130 135 140

Asn Asp Tyr Leu Gly His Ser Ser Phe Ile Leu Pro Pro Leu Pro AsnAsn Asp Tyr Leu Gly His Ser Ser Phe Ile Leu Pro Pro Leu Pro Asn

145 150 155 160145 150 155 160

Thr Gln Phe Thr Arg Asp Thr Thr Cys Trp Ile Tyr Gly Gly Val ThrThr Gln Phe Thr Arg Asp Thr Thr Cys Trp Ile Tyr Gly Gly Val Thr

165 170 175 165 170 175

Leu Asn Pro Met Tyr Trp Pro Ala Arg Arg Gln Glu Thr Leu Leu ThrLeu Asn Pro Met Tyr Trp Pro Ala Arg Arg Gln Glu Thr Leu Leu Thr

180 185 190 180 185 190

Thr Ala Ile Tyr Lys Phe His Pro Gln Phe Thr Gly Ala Asp Phe GlnThr Ala Ile Tyr Lys Phe His Pro Gln Phe Thr Gly Ala Asp Phe Gln

195 200 205 195 200 205

Val Trp Tyr Gly Asp Pro Asp Lys Asp His Gly Asn Ala Thr Leu GluVal Trp Tyr Gly Asp Pro Asp Lys Asp His Gly Asn Ala Thr Leu Glu

210 215 220 210 215 220

Gly Gly Asp Val Met Pro Ile Gly Lys Gly Ile Val Leu Ile Gly MetGly Gly Asp Val Met Pro Ile Gly Lys Gly Ile Val Leu Ile Gly Met

225 230 235 240225 230 235 240

Gly Glu Arg Thr Ser Arg Gln Ala Ile Gly Gln Leu Ala Gln Asn LeuGly Glu Arg Thr Ser Arg Gln Ala Ile Gly Gln Leu Ala Gln Asn Leu

245 250 255 245 250 255

Phe Ala Lys Gly Ala Val Glu Lys Val Ile Val Ala Gly Leu Pro LysPhe Ala Lys Gly Ala Val Glu Lys Val Ile Val Ala Gly Leu Pro Lys

260 265 270 260 265 270

Ser Arg Ala Ala Met His Leu Asp Thr Val Phe Ser Phe Cys Asp ArgSer Arg Ala Ala Met His Leu Asp Thr Val Phe Ser Phe Cys Asp Arg

275 280 285 275 280 285

Asp Leu Val Thr Ile Phe Pro Glu Val Val Lys Glu Ile Val Pro PheAsp Leu Val Thr Ile Phe Pro Glu Val Val Lys Glu Ile Val Pro Phe

290 295 300 290 295 300

Ile Ile Arg Pro Asp Glu Ser Lys Pro Tyr Gly Met Asp Val Arg ArgIle Ile Arg Pro Asp Glu Ser Lys Pro Tyr Gly Met Asp Val Arg Arg

305 310 315 320305 310 315 320

Glu Asn Lys Ser Phe Ile Glu Val Val Gly Glu Gln Leu Gly Val LysGlu Asn Lys Ser Phe Ile Glu Val Val Gly Glu Gln Leu Gly Val Lys

325 330 335 325 330 335

Leu Arg Val Val Glu Thr Gly Gly Asn Ser Phe Ala Ala Glu Arg GluLeu Arg Val Val Glu Thr Gly Gly Asn Ser Phe Ala Ala Glu Arg Glu

340 345 350 340 345 350

Gln Trp Asp Asp Gly Asn Asn Val Val Ala Val Glu Pro Gly Val ValGln Trp Asp Asp Gly Asn Asn Val Val Ala Val Glu Pro Gly Val Val

355 360 365 355 360 365

Ile Gly Tyr Asp Arg Asn Thr Tyr Thr Asn Thr Leu Leu Arg Lys AlaIle Gly Tyr Asp Arg Asn Thr Tyr Thr Asn Thr Leu Leu Arg Lys Ala

370 375 380 370 375 380

Gly Ile Glu Val Ile Thr Ile Ser Ala Gly Glu Leu Gly Arg Gly ArgGly Ile Glu Val Ile Thr Ile Ser Ala Gly Glu Leu Gly Arg Gly Arg

385 390 395 400385 390 395 400

Gly Gly Gly His Cys Met Thr Cys Pro Ile Val Arg Asp Pro Ile AspGly Gly Gly His Cys Met Thr Cys Pro Ile Val Arg Asp Pro Ile Asp

405 410 415 405 410 415

TyrTyr

<210> 25<210> 25

<211> 418<211> 418

<212> PRT<212> PRT

<213> 铜绿假单孢菌（Pseudomonas aeruginosa）<213> Pseudomonas aeruginosa

<400> 25<400> 25

Met Ser Thr Glu Lys Thr Lys Leu Gly Val His Ser Glu Ala Gly LysMet Ser Thr Glu Lys Thr Lys Leu Gly Val His Ser Glu Ala Gly Lys

1 5 10 151 5 10 15

Leu Arg Lys Val Met Val Cys Ser Pro Gly Leu Ala His Gln Arg LeuLeu Arg Lys Val Met Val Cys Ser Pro Gly Leu Ala His Gln Arg Leu

20 25 30 20 25 30

Thr Pro Ser Asn Cys Asp Glu Leu Leu Phe Asp Asp Val Ile Trp ValThr Pro Ser Asn Cys Asp Glu Leu Leu Phe Asp Asp Val Ile Trp Val

35 40 45 35 40 45

Asn Gln Ala Lys Arg Asp His Phe Asp Phe Val Thr Lys Met Arg GluAsn Gln Ala Lys Arg Asp His Phe Asp Phe Val Thr Lys Met Arg Glu

50 55 60 50 55 60

Arg Gly Ile Asp Val Leu Glu Met His Asn Leu Leu Thr Glu Thr IleArg Gly Ile Asp Val Leu Glu Met His Asn Leu Leu Thr Glu Thr Ile

65 70 75 8065 70 75 80

Gln Asn Pro Glu Ala Leu Lys Trp Ile Leu Asp Arg Lys Ile Thr AlaGln Asn Pro Glu Ala Leu Lys Trp Ile Leu Asp Arg Lys Ile Thr Ala

85 90 95 85 90 95

Asp Ser Val Gly Leu Gly Leu Thr Ser Glu Leu Arg Ser Trp Leu GluAsp Ser Val Gly Leu Gly Leu Thr Ser Glu Leu Arg Ser Trp Leu Glu

100 105 110 100 105 110

Ser Leu Glu Pro Arg Lys Leu Ala Glu Tyr Leu Ile Gly Gly Val AlaSer Leu Glu Pro Arg Lys Leu Ala Glu Tyr Leu Ile Gly Gly Val Ala

115 120 125 115 120 125

Ala Asp Asp Leu Pro Ala Ser Glu Gly Ala Asn Ile Leu Lys Met TyrAla Asp Asp Leu Pro Ala Ser Glu Gly Ala Asn Ile Leu Lys Met Tyr

130 135 140 130 135 140

Arg Glu Tyr Leu Gly His Ser Ser Phe Leu Leu Pro Pro Leu Pro AsnArg Glu Tyr Leu Gly His Ser Ser Phe Leu Leu Pro Pro Leu Pro Asn

145 150 155 160145 150 155 160

Thr Gln Phe Thr Arg Asp Thr Thr Cys Trp Ile Tyr Gly Gly Val ThrThr Gln Phe Thr Arg Asp Thr Thr Cys Trp Ile Tyr Gly Gly Val Thr

165 170 175 165 170 175

Leu Asn Pro Met Tyr Trp Pro Ala Arg Arg Gln Glu Thr Leu Leu ThrLeu Asn Pro Met Tyr Trp Pro Ala Arg Arg Gln Glu Thr Leu Leu Thr

180 185 190 180 185 190

Thr Ala Ile Tyr Lys Phe His Pro Glu Phe Ala Asn Ala Glu Phe GluThr Ala Ile Tyr Lys Phe His Pro Glu Phe Ala Asn Ala Glu Phe Glu

195 200 205 195 200 205

Ile Trp Tyr Gly Asp Pro Asp Lys Asp His Gly Ser Ser Thr Leu GluIle Trp Tyr Gly Asp Pro Asp Lys Asp His Gly Ser Ser Thr Leu Glu

210 215 220 210 215 220

Gly Gly Asp Val Met Pro Ile Gly Asn Gly Val Val Leu Ile Gly MetGly Gly Asp Val Met Pro Ile Gly Asn Gly Val Val Leu Ile Gly Met

225 230 235 240225 230 235 240

Gly Glu Arg Ser Ser Arg Gln Ala Ile Gly Gln Val Ala Gln Ser LeuGly Glu Arg Ser Ser Arg Gln Ala Ile Gly Gln Val Ala Gln Ser Leu

245 250 255 245 250 255

Phe Ala Lys Gly Ala Ala Glu Arg Val Ile Val Ala Gly Leu Pro LysPhe Ala Lys Gly Ala Ala Glu Arg Val Ile Val Ala Gly Leu Pro Lys

260 265 270 260 265 270

Ser Arg Ala Ala Met His Leu Asp Thr Val Phe Ser Phe Cys Asp ArgSer Arg Ala Ala Met His Leu Asp Thr Val Phe Ser Phe Cys Asp Arg

275 280 285 275 280 285

Asp Leu Val Thr Val Phe Pro Glu Val Val Lys Glu Ile Val Pro PheAsp Leu Val Thr Val Phe Pro Glu Val Val Lys Glu Ile Val Pro Phe

290 295 300 290 295 300

Ser Leu Arg Pro Asp Ala Ser Ser Pro Tyr Gly Met Ser Ile Arg ArgSer Leu Arg Pro Asp Ala Ser Ser Pro Tyr Gly Met Ser Ile Arg Arg

305 310 315 320305 310 315 320

Glu Glu Lys Thr Phe Leu Glu Val Val Ala Glu Ser Leu Gly Leu LysGlu Glu Lys Thr Phe Leu Glu Val Val Ala Glu Ser Leu Gly Leu Lys

325 330 335 325 330 335

Lys Leu Arg Val Val Glu Thr Gly Gly Asn Ser Phe Ala Ala Glu ArgLys Leu Arg Val Val Glu Thr Gly Gly Asn Ser Phe Ala Ala Glu Arg

340 345 350 340 345 350

Glu Gln Trp Asp Asp Gly Asn Asn Val Val Cys Leu Glu Pro Gly ValGlu Gln Trp Asp Asp Gly Asn Asn Val Val Cys Leu Glu Pro Gly Val

355 360 365 355 360 365

Val Val Gly Tyr Asp Arg Asn Thr Tyr Thr Asn Thr Leu Leu Arg LysVal Val Gly Tyr Asp Arg Asn Thr Tyr Thr Asn Thr Leu Leu Arg Lys

370 375 380 370 375 380

Ala Gly Val Glu Val Ile Thr Ile Ser Ala Ser Glu Leu Gly Arg GlyAla Gly Val Glu Val Ile Thr Ile Ser Ala Ser Glu Leu Gly Arg Gly

385 390 395 400385 390 395 400

Arg Gly Gly Gly His Cys Met Thr Cys Pro Ile Ile Arg Asp Pro IleArg Gly Gly Gly His Cys Met Thr Cys Pro Ile Ile Arg Asp Pro Ile

405 410 415 405 410 415

Asp TyrAsp Tyr

<210> 26<210> 26

<211> 402<211> 402

<212> PRT<212> PRT

<213> 结核分枝杆菌群（Mycobacterium tuberculosis complex）<213> Mycobacterium tuberculosis complex

<400> 26<400> 26

Met Gly Val Glu Leu Gly Ser Asn Ser Glu Val Gly Ala Leu Arg ValMet Gly Val Glu Leu Gly Ser Asn Ser Glu Val Gly Ala Leu Arg Val

1 5 10 151 5 10 15

Val Ile Leu His Arg Pro Gly Ala Glu Leu Arg Arg Leu Thr Pro ArgVal Ile Leu His Arg Pro Gly Ala Glu Leu Arg Arg Leu Thr Pro Arg

20 25 30 20 25 30

Asn Thr Asp Gln Leu Leu Phe Asp Gly Leu Pro Trp Val Ser Arg AlaAsn Thr Asp Gln Leu Leu Phe Asp Gly Leu Pro Trp Val Ser Arg Ala

35 40 45 35 40 45

Gln Asp Glu His Asp Glu Phe Ala Glu Leu Leu Ala Ser Arg Gly AlaGln Asp Glu His Asp Glu Phe Ala Glu Leu Leu Ala Ser Arg Gly Ala

50 55 60 50 55 60

Glu Val Leu Leu Leu Ser Asp Leu Leu Thr Glu Ala Leu His His SerGlu Val Leu Leu Leu Ser Asp Leu Leu Thr Glu Ala Leu His His Ser

65 70 75 8065 70 75 80

Gly Ala Ala Arg Met Gln Gly Ile Ala Ala Ala Val Asp Ala Pro ArgGly Ala Ala Arg Met Gln Gly Ile Ala Ala Ala Val Asp Ala Pro Arg

85 90 95 85 90 95

Leu Gly Leu Pro Leu Ala Gln Glu Leu Ser Ala Tyr Leu Arg Ser LeuLeu Gly Leu Pro Leu Ala Gln Glu Leu Ser Ala Tyr Leu Arg Ser Leu

100 105 110 100 105 110

Asp Pro Gly Arg Leu Ala His Val Leu Thr Ala Gly Met Thr Phe AsnAsp Pro Gly Arg Leu Ala His Val Leu Thr Ala Gly Met Thr Phe Asn

115 120 125 115 120 125

Glu Leu Pro Ser Asp Thr Arg Thr Asp Val Ser Leu Val Leu Arg MetGlu Leu Pro Ser Asp Thr Arg Thr Asp Val Ser Leu Val Leu Arg Met

130 135 140 130 135 140

His His Gly Gly Asp Phe Val Ile Glu Pro Leu Pro Asn Leu Val PheHis His Gly Gly Asp Phe Val Ile Glu Pro Leu Pro Asn Leu Val Phe

145 150 155 160145 150 155 160

Thr Arg Asp Ser Ser Ile Trp Ile Gly Pro Arg Val Val Ile Pro SerThr Arg Asp Ser Ser Ile Trp Ile Gly Pro Arg Val Val Ile Pro Ser

165 170 175 165 170 175

Leu Ala Leu Arg Ala Arg Val Arg Glu Ala Ser Leu Thr Asp Leu IleLeu Ala Leu Arg Ala Arg Val Arg Glu Ala Ser Leu Thr Asp Leu Ile

180 185 190 180 185 190

Tyr Ala His His Pro Arg Phe Thr Gly Val Arg Arg Ala Tyr Glu SerTyr Ala His His Pro Arg Phe Thr Gly Val Arg Arg Ala Tyr Glu Ser

195 200 205 195 200 205

Arg Thr Ala Pro Val Glu Gly Gly Asp Val Leu Leu Leu Ala Pro GlyArg Thr Ala Pro Val Glu Gly Gly Asp Val Leu Leu Leu Ala Pro Gly

210 215 220 210 215 220

Val Val Ala Val Gly Val Gly Glu Arg Thr Thr Pro Ala Gly Ala GluVal Val Ala Val Gly Val Gly Glu Arg Thr Thr Pro Ala Gly Ala Glu

225 230 235 240225 230 235 240

Ala Leu Ala Arg Ser Leu Phe Asp Asp Asp Leu Ala His Thr Val LeuAla Leu Ala Arg Ser Leu Phe Asp Asp Asp Leu Ala His Thr Val Leu

245 250 255 245 250 255

Ala Val Pro Ile Ala Gln Gln Arg Ala Gln Met His Leu Asp Thr ValAla Val Pro Ile Ala Gln Gln Arg Ala Gln Met His Leu Asp Thr Val

260 265 270 260 265 270

Cys Thr Met Val Asp Thr Asp Thr Met Val Met Tyr Ala Asn Val ValCys Thr Met Val Asp Thr Asp Thr Met Val Met Tyr Ala Asn Val Val

275 280 285 275 280 285

Asp Thr Leu Glu Ala Phe Thr Ile Gln Arg Thr Pro Asp Gly Val ThrAsp Thr Leu Glu Ala Phe Thr Ile Gln Arg Thr Pro Asp Gly Val Thr

290 295 300 290 295 300

Ile Gly Asp Ala Ala Pro Phe Ala Glu Ala Ala Ala Lys Ala Met GlyIle Gly Asp Ala Ala Pro Phe Ala Glu Ala Ala Ala Lys Ala Met Gly

305 310 315 320305 310 315 320

Ile Asp Lys Leu Arg Val Ile His Thr Gly Met Asp Pro Val Val AlaIle Asp Lys Leu Arg Val Ile His Thr Gly Met Asp Pro Val Val Ala

325 330 335 325 330 335

Glu Arg Glu Gln Trp Asp Asp Gly Asn Asn Thr Leu Ala Leu Ala ProGlu Arg Glu Gln Trp Asp Asp Gly Asn Asn Thr Leu Ala Leu Ala Pro

340 345 350 340 345 350

Gly Val Val Val Ala Tyr Glu Arg Asn Val Gln Thr Asn Ala Arg LeuGly Val Val Val Ala Tyr Glu Arg Asn Val Gln Thr Asn Ala Arg Leu

355 360 365 355 360 365

Gln Asp Ala Gly Ile Glu Val Leu Thr Ile Ala Gly Ser Glu Leu GlyGln Asp Ala Gly Ile Glu Val Leu Thr Ile Ala Gly Ser Glu Leu Gly

370 375 380 370 375 380

Thr Gly Arg Gly Gly Pro Arg Cys Met Ser Cys Pro Ala Ala Arg AspThr Gly Arg Gly Gly Pro Arg Cys Met Ser Cys Pro Ala Ala Arg Asp

385 390 395 400385 390 395 400

Pro LeuPro Leu

<210> 27<210> 27

<211> 409<211> 409

<212> PRT<212> PRT

<213> 关节炎支原体（Mycoplasma arthritidis）<213> Mycoplasma arthritidis

<400> 27<400> 27

Met Ser Val Phe Asp Ser Lys Phe Lys Gly Ile His Val Tyr Ser GluMet Ser Val Phe Asp Ser Lys Phe Lys Gly Ile His Val Tyr Ser Glu

1 5 10 151 5 10 15

Ile Gly Glu Leu Glu Thr Val Leu Val His Glu Pro Gly Lys Glu IleIle Gly Glu Leu Glu Thr Val Leu Val His Glu Pro Gly Lys Glu Ile

20 25 30 20 25 30

Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala IleAsp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile

35 40 45 35 40 45

Leu Glu Ser His Asp Ala Arg Lys Glu His Lys Glu Phe Val Ala GluLeu Glu Ser His Asp Ala Arg Lys Glu His Lys Glu Phe Val Ala Glu

50 55 60 50 55 60

Leu Lys Lys Arg Gly Ile Asn Val Val Glu Leu Val Asp Leu Ile ValLeu Lys Lys Arg Gly Ile Asn Val Val Glu Leu Val Asp Leu Ile Val

65 70 75 8065 70 75 80

Glu Thr Tyr Asp Leu Ala Ser Lys Glu Ala Lys Glu Lys Leu Leu GluGlu Thr Tyr Asp Leu Ala Ser Lys Glu Ala Lys Glu Lys Leu Leu Glu

85 90 95 85 90 95

Glu Phe Leu Asp Asp Ser Val Pro Val Leu Ser Asp Glu His Arg AlaGlu Phe Leu Asp Asp Ser Val Pro Val Leu Ser Asp Glu His Arg Ala

100 105 110 100 105 110

Ala Val Lys Lys Phe Leu Gln Ser Gln Lys Ser Thr Arg Ser Leu ValAla Val Lys Lys Phe Leu Gln Ser Gln Lys Ser Thr Arg Ser Leu Val

115 120 125 115 120 125

Glu Tyr Met Ile Ala Gly Ile Thr Lys His Asp Leu Lys Ile Glu SerGlu Tyr Met Ile Ala Gly Ile Thr Lys His Asp Leu Lys Ile Glu Ser

130 135 140 130 135 140

Asp Leu Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr ArgAsp Leu Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg

145 150 155 160145 150 155 160

Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met ArgAsp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg

165 170 175 165 170 175

Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Val Phe SerTyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Val Phe Ser

180 185 190 180 185 190

Asn His Pro Lys Leu Val Asn Thr Pro Trp Tyr Tyr Asp Pro Ala GluAsn His Pro Lys Leu Val Asn Thr Pro Trp Tyr Tyr Asp Pro Ala Glu

195 200 205 195 200 205

Gly Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp ThrGly Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr

210 215 220 210 215 220

Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Gln Thr Ile Thr LeuLeu Val Val Gly Val Ser Glu Arg Thr Asp Leu Gln Thr Ile Thr Leu

225 230 235 240225 230 235 240

Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu Cys Glu Phe Lys Arg IleLeu Ala Lys Asn Ile Lys Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile

245 250 255 245 250 255

Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp ThrVal Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr

260 265 270 260 265 270

Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile AlaTrp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala

275 280 285 275 280 285

Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly AspAsn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Asp

290 295 300 290 295 300

Ala Pro Gln Pro Val Asp Asn Gly Leu Pro Leu Glu Asp Leu Leu LysAla Pro Gln Pro Val Asp Asn Gly Leu Pro Leu Glu Asp Leu Leu Lys

305 310 315 320305 310 315 320

Ser Ile Ile Gly Lys Lys Pro Thr Leu Ile Pro Ile Ala Gly Ala GlySer Ile Ile Gly Lys Lys Pro Thr Leu Ile Pro Ile Ala Gly Ala Gly

325 330 335 325 330 335

Ala Ser Gln Ile Asp Ile Glu Arg Glu Thr His Phe Asp Gly Thr AsnAla Ser Gln Ile Asp Ile Glu Arg Glu Thr His Phe Asp Gly Thr Asn

340 345 350 340 345 350

Tyr Leu Ala Val Ala Pro Gly Ile Val Ile Gly Tyr Ala Arg Asn GluTyr Leu Ala Val Ala Pro Gly Ile Val Ile Gly Tyr Ala Arg Asn Glu

355 360 365 355 360 365

Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Thr Val Leu Pro PheLys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Thr Val Leu Pro Phe

370 375 380 370 375 380

Arg Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met SerArg Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser

385 390 395 400385 390 395 400

Met Pro Leu Ser Arg Lys Asp Val LysMet Pro Leu Ser Arg Lys Asp Val Lys

405 405

<210> 28<210> 28

<211> 410<211> 410

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 海豹脑支原体人工全长<223> Seal artificial full length of Mycoplasma cerebrum

<400> 28<400> 28

Met Ser Val Phe Asp Ser Lys Phe Asn Gly Ile His Val Tyr Ser GluMet Ser Val Phe Asp Ser Lys Phe Asn Gly Ile His Val Tyr Ser Glu

1 5 10 151 5 10 15

Ile Gly Glu Leu Glu Thr Val Leu Val His Glu Pro Gly Arg Glu IleIle Gly Glu Leu Glu Thr Val Leu Val His Glu Pro Gly Arg Glu Ile

20 25 30 20 25 30

Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala IleAsp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile

35 40 45 35 40 45

Leu Glu Ser His Asp Ala Arg Lys Glu His Gln Ser Phe Val Lys GlnLeu Glu Ser His Asp Ala Arg Lys Glu His Gln Ser Phe Val Lys Gln

50 55 60 50 55 60

Leu Lys Asp Asn Gly Ile Asn Val Val Glu Leu Thr Asp Leu Val AlaLeu Lys Asp Asn Gly Ile Asn Val Val Glu Leu Thr Asp Leu Val Ala

65 70 75 8065 70 75 80

Glu Thr Phe Asp Leu Ala Ser Lys Glu Glu Gln Glu Lys Leu Ile GluGlu Thr Phe Asp Leu Ala Ser Lys Glu Glu Gln Glu Lys Leu Ile Glu

85 90 95 85 90 95

Glu Phe Leu Glu Asp Ser Glu Pro Val Leu Ser Glu Ala His Lys ThrGlu Phe Leu Glu Asp Ser Glu Pro Val Leu Ser Glu Ala His Lys Thr

100 105 110 100 105 110

Ala Val Arg Lys Phe Leu Thr Ser Arg Lys Ser Thr Arg Glu Met ValAla Val Arg Lys Phe Leu Thr Ser Arg Lys Ser Thr Arg Glu Met Val

115 120 125 115 120 125

Glu Phe Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Glu AlaGlu Phe Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Glu Ala

130 135 140 130 135 140

Asp His Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr ArgAsp His Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg

145 150 155 160145 150 155 160

Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met ArgAsp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg

165 170 175 165 170 175

Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Val Phe SerTyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Val Phe Ser

180 185 190 180 185 190

Asn His Pro Lys Leu Val Lys Thr Pro Trp Tyr Tyr Asp Pro Ala MetAsn His Pro Lys Leu Val Lys Thr Pro Trp Tyr Tyr Asp Pro Ala Met

195 200 205 195 200 205

Lys Met Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp ThrLys Met Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr

210 215 220 210 215 220

Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Glu Thr Ile Thr LeuLeu Val Val Gly Val Ser Glu Arg Thr Asp Leu Glu Thr Ile Thr Leu

225 230 235 240225 230 235 240

Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu Val Glu Phe Lys Arg IleLeu Ala Lys Asn Ile Lys Ala Asn Lys Glu Val Glu Phe Lys Arg Ile

245 250 255 245 250 255

Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp ThrVal Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr

260 265 270 260 265 270

Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile AlaTrp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala

275 280 285 275 280 285

Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly AlaAsn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ala

290 295 300 290 295 300

Glu Pro Gln Pro Lys Glu Asn Gly Leu Pro Leu Glu Gly Leu Leu GlnGlu Pro Gln Pro Lys Glu Asn Gly Leu Pro Leu Glu Gly Leu Leu Gln

305 310 315 320305 310 315 320

Ser Ile Ile Asn Lys Lys Pro Val Leu Ile Pro Ile Ala Gly Asn AsnSer Ile Ile Asn Lys Lys Pro Val Leu Ile Pro Ile Ala Gly Asn Asn

325 330 335 325 330 335

Ala Ser His Ile Asp Ile Glu Arg Glu Thr His Phe Asp Gly Thr AsnAla Ser His Ile Asp Ile Glu Arg Glu Thr His Phe Asp Gly Thr Asn

340 345 350 340 345 350

Tyr Leu Ala Ile Lys Pro Gly Val Val Ile Gly Tyr Ala Arg Asn GluTyr Leu Ala Ile Lys Pro Gly Val Val Ile Gly Tyr Ala Arg Asn Glu

355 360 365 355 360 365

Lys Thr Asn Ala Ala Leu Ala Ala Ala Gly Ile Lys Val Leu Pro PheLys Thr Asn Ala Ala Leu Ala Ala Ala Gly Ile Lys Val Leu Pro Phe

370 375 380 370 375 380

His Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met SerHis Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser

385 390 395 400385 390 395 400

Met Pro Leu Ser Arg Lys Asp Val Lys TrpMet Pro Leu Ser Arg Lys Asp Val Lys Trp

405 410 405 410

<210> 29<210> 29

<211> 410<211> 410

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 猫支原体人工全长<223> Mycoplasma felis artificial full length

<400> 29<400> 29

Met Ser Val Phe Asp Ser Lys Phe Asn Gly Ile His Val Tyr Ser GluMet Ser Val Phe Asp Ser Lys Phe Asn Gly Ile His Val Tyr Ser Glu

1 5 10 151 5 10 15

Ile Gly Glu Leu Glu Ser Val Leu Val His Glu Pro Gly Arg Glu IleIle Gly Glu Leu Glu Ser Val Leu Val His Glu Pro Gly Arg Glu Ile

20 25 30 20 25 30

Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala IleAsp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile

35 40 45 35 40 45

Leu Glu Ser His Asp Ala Arg Lys Glu His Lys Leu Phe Val Ser GluLeu Glu Ser His Asp Ala Arg Lys Glu His Lys Leu Phe Val Ser Glu

50 55 60 50 55 60

Leu Lys Ala Asn Asp Ile Asn Val Val Glu Leu Thr Asp Leu Val ThrLeu Lys Ala Asn Asp Ile Asn Val Val Glu Leu Thr Asp Leu Val Thr

65 70 75 8065 70 75 80

Glu Thr Tyr Asp Leu Ala Ser Gln Glu Ala Lys Asp Asn Leu Ile GluGlu Thr Tyr Asp Leu Ala Ser Gln Glu Ala Lys Asp Asn Leu Ile Glu

85 90 95 85 90 95

Glu Phe Leu Glu Asp Ser Glu Pro Val Leu Thr Glu Glu Leu Lys SerGlu Phe Leu Glu Asp Ser Glu Pro Val Leu Thr Glu Glu Leu Lys Ser

100 105 110 100 105 110

Val Val Arg Thr Tyr Leu Lys Ser Ile Lys Ser Thr Arg Glu Leu IleVal Val Arg Thr Tyr Leu Lys Ser Ile Lys Ser Thr Arg Glu Leu Ile

115 120 125 115 120 125

Gln Met Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Glu AlaGln Met Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Glu Ala

130 135 140 130 135 140

Asp His Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr ArgAsp His Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg

145 150 155 160145 150 155 160

Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met ArgAsp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg

165 170 175 165 170 175

Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Val Phe SerTyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Val Phe Ser

180 185 190 180 185 190

Asn His Pro Lys Leu Val Asn Thr Pro Trp Tyr Tyr Asp Pro Ser LeuAsn His Pro Lys Leu Val Asn Thr Pro Trp Tyr Tyr Asp Pro Ser Leu

195 200 205 195 200 205

Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asn ThrLys Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asn Thr

210 215 220 210 215 220

Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Glu Thr Val Thr LeuLeu Val Val Gly Val Ser Glu Arg Thr Asp Leu Glu Thr Val Thr Leu

225 230 235 240225 230 235 240

Leu Ala Lys Asn Ile Val Ala Asn Lys Glu Cys Glu Phe Lys Arg IleLeu Ala Lys Asn Ile Val Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile

245 250 255 245 250 255

Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp ThrVal Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr

260 265 270 260 265 270

Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile AlaTrp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala

275 280 285 275 280 285

Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly GluAsn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Glu

290 295 300 290 295 300

Glu Pro Gln Pro Val Glu Asn Gly Leu Pro Leu Glu Gly Leu Leu GluGlu Pro Gln Pro Val Glu Asn Gly Leu Pro Leu Glu Gly Leu Leu Glu

305 310 315 320305 310 315 320

Ser Ile Ile Asn Lys Lys Pro Ile Leu Ile Pro Ile Ala Gly Glu GlySer Ile Ile Asn Lys Lys Pro Ile Leu Ile Pro Ile Ala Gly Glu Gly

325 330 335 325 330 335

Ala Ser Gln Ile Asp Ile Glu Arg Glu Thr His Phe Asp Gly Thr AsnAla Ser Gln Ile Asp Ile Glu Arg Glu Thr His Phe Asp Gly Thr Asn

340 345 350 340 345 350

Tyr Leu Ala Ile Arg Pro Gly Val Val Ile Gly Tyr Ser Arg Asn GluTyr Leu Ala Ile Arg Pro Gly Val Val Ile Gly Tyr Ser Arg Asn Glu

355 360 365 355 360 365

Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Lys Val Leu Pro PheLys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Lys Val Leu Pro Phe

370 375 380 370 375 380

His Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met SerHis Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser

385 390 395 400385 390 395 400

Met Pro Leu Ser Arg Lys Asp Val Lys TrpMet Pro Leu Ser Arg Lys Asp Val Lys Trp

405 410 405 410

<210> 30<210> 30

<211> 410<211> 410

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 海豹支原体人工全长<223> Artificial Full Length of Mycoplasma Seals

<400> 30<400> 30

Met Ser Val Phe Asp Ser Lys Phe Asn Gly Ile His Val Tyr Ser GluMet Ser Val Phe Asp Ser Lys Phe Asn Gly Ile His Val Tyr Ser Glu

1 5 10 151 5 10 15

Ile Gly Glu Leu Gln Thr Val Leu Val His Glu Pro Gly Arg Glu IleIle Gly Glu Leu Gln Thr Val Leu Val His Glu Pro Gly Arg Glu Ile

20 25 30 20 25 30

Glu Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala IleGlu Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile

35 40 45 35 40 45

Leu Glu Ser His Asp Ala Arg Lys Glu His Gln Glu Phe Val Ala GluLeu Glu Ser His Asp Ala Arg Lys Glu His Gln Glu Phe Val Ala Glu

50 55 60 50 55 60

Leu Lys Lys Asn Asn Ile Asn Val Val Glu Leu Thr Asp Leu Val SerLeu Lys Lys Asn Asn Ile Asn Val Val Glu Leu Thr Asp Leu Val Ser

65 70 75 8065 70 75 80

Glu Thr Tyr Asp Met Val Ser Lys Glu Lys Gln Glu Lys Leu Ile GluGlu Thr Tyr Asp Met Val Ser Lys Glu Lys Gln Glu Lys Leu Ile Glu

85 90 95 85 90 95

Glu Phe Leu Glu Asp Ser Glu Pro Val Leu Ser Glu Glu His Lys GlyGlu Phe Leu Glu Asp Ser Glu Pro Val Leu Ser Glu Glu His Lys Gly

100 105 110 100 105 110

Leu Val Arg Lys Phe Leu Lys Ser Leu Lys Ser Ser Lys Glu Leu IleLeu Val Arg Lys Phe Leu Lys Ser Leu Lys Ser Ser Lys Glu Leu Ile

115 120 125 115 120 125

Gln Tyr Met Met Ala Gly Ile Thr Lys His Asp Leu Asn Ile Glu AlaGln Tyr Met Met Ala Gly Ile Thr Lys His Asp Leu Asn Ile Glu Ala

130 135 140 130 135 140

Asp His Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr ArgAsp His Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg

145 150 155 160145 150 155 160

Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met ArgAsp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg

165 170 175 165 170 175

Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Ile Phe AlaTyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Ile Phe Ala

180 185 190 180 185 190

Asn His Pro Lys Leu Met Asn Thr Pro Leu Tyr Tyr Asn Pro Asp MetAsn His Pro Lys Leu Met Asn Thr Pro Leu Tyr Tyr Asn Pro Asp Met

195 200 205 195 200 205

Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Val Tyr Asn Asn Glu ThrLys Leu Ser Ile Glu Gly Gly Asp Val Phe Val Tyr Asn Asn Glu Thr

210 215 220 210 215 220

Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Asp Thr Ile Thr LeuLeu Val Val Gly Val Ser Glu Arg Thr Asp Leu Asp Thr Ile Thr Leu

225 230 235 240225 230 235 240

Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu Arg Glu Phe Lys Arg IleLeu Ala Lys Asn Ile Lys Ala Asn Lys Glu Arg Glu Phe Lys Arg Ile

245 250 255 245 250 255

Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp ThrVal Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr

260 265 270 260 265 270

Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile AlaTrp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala

275 280 285 275 280 285

Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly AspAsn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Asp

290 295 300 290 295 300

Glu Pro Gln Pro Lys Val Asn Gly Leu Pro Leu Glu Lys Leu Leu GluGlu Pro Gln Pro Lys Val Asn Gly Leu Pro Leu Glu Lys Leu Leu Glu

305 310 315 320305 310 315 320

Ser Ile Ile Asn Lys Lys Pro Ile Leu Ile Pro Ile Ala Gly Thr SerSer Ile Ile Asn Lys Lys Pro Ile Leu Ile Pro Ile Ala Gly Thr Ser

325 330 335 325 330 335

Ala Ser Asn Ile Asp Val Glu Arg Glu Thr His Phe Asp Gly Thr AsnAla Ser Asn Ile Asp Val Glu Arg Glu Thr His Phe Asp Gly Thr Asn

340 345 350 340 345 350

Tyr Leu Ala Ile Ala Pro Gly Val Val Ile Gly Tyr Ser Arg Asn ValTyr Leu Ala Ile Ala Pro Gly Val Val Ile Gly Tyr Ser Arg Asn Val

355 360 365 355 360 365

Lys Thr Asn Glu Ala Leu Glu Ala Ala Gly Ile Lys Val Leu Pro PheLys Thr Asn Glu Ala Leu Glu Ala Ala Gly Ile Lys Val Leu Pro Phe

370 375 380 370 375 380

Lys Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met SerLys Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser

385 390 395 400385 390 395 400

Met Pro Leu Ser Arg Lys Asp Val Lys TrpMet Pro Leu Ser Arg Lys Asp Val Lys Trp

405 410 405 410

<210> 31<210> 31

<211> 410<211> 410

<212> PRT<212> PRT

<213> 唾液支原体（Mycoplasma salivarium）<213> Mycoplasma salivarium

<400> 31<400> 31

Met Ser Val Phe Ser Ser Lys Phe Asn Gly Ile His Val Tyr Ser GluMet Ser Val Phe Ser Ser Lys Phe Asn Gly Ile His Val Tyr Ser Glu

1 5 10 151 5 10 15

Ile Gly Glu Leu Glu Thr Val Leu Val His Glu Pro Gly Lys Glu IleIle Gly Glu Leu Glu Thr Val Leu Val His Glu Pro Gly Lys Glu Ile

20 25 30 20 25 30

Asp Tyr Ile Thr Pro Ser Arg Leu Asp Glu Leu Leu Phe Ser Ala IleAsp Tyr Ile Thr Pro Ser Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile

35 40 45 35 40 45

Leu Glu Ser His Asp Ala Arg Lys Glu His Gln Glu Phe Val Ala ThrLeu Glu Ser His Asp Ala Arg Lys Glu His Gln Glu Phe Val Ala Thr

50 55 60 50 55 60

Leu Lys Lys Glu Lys Ile Asn Val Val Glu Leu Thr Asp Leu Val ThrLeu Lys Lys Glu Lys Ile Asn Val Val Glu Leu Thr Asp Leu Val Thr

65 70 75 8065 70 75 80

Glu Thr Tyr Asp Leu Val Asp Gln Lys Thr Lys Asp Lys Leu Ile AspGlu Thr Tyr Asp Leu Val Asp Gln Lys Thr Lys Asp Lys Leu Ile Asp

85 90 95 85 90 95

Glu Phe Leu Glu Asp Ser Glu Pro Val Leu Thr Ala Glu Leu Lys AlaGlu Phe Leu Glu Asp Ser Glu Pro Val Leu Thr Ala Glu Leu Lys Ala

100 105 110 100 105 110

Thr Val Lys Lys Phe Leu Lys Ser Phe Lys Glu Thr Arg Lys Leu IleThr Val Lys Lys Phe Leu Lys Ser Phe Lys Glu Thr Arg Lys Leu Ile

115 120 125 115 120 125

Glu Val Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Lys AlaGlu Val Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Lys Ala

130 135 140 130 135 140

Asp Arg Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr ArgAsp Arg Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg

145 150 155 160145 150 155 160

Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met ArgAsp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg

165 170 175 165 170 175

Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Ile Phe AsnTyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Ile Phe Asn

180 185 190 180 185 190

Asn His Pro Lys Leu Val Lys Thr Pro Trp Tyr Tyr Asp Pro Ala MetAsn His Pro Lys Leu Val Lys Thr Pro Trp Tyr Tyr Asp Pro Ala Met

195 200 205 195 200 205

Lys Met Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp ThrLys Met Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr

210 215 220 210 215 220

Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Asp Thr Ile Thr LeuLeu Val Val Gly Val Ser Glu Arg Thr Asp Leu Asp Thr Ile Thr Leu

225 230 235 240225 230 235 240

Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu Cys Glu Phe Lys Arg IleLeu Ala Lys Asn Ile Lys Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile

245 250 255 245 250 255

Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp ThrVal Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr

260 265 270 260 265 270

Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile AlaTrp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala

275 280 285 275 280 285

Asn Asp Ile Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly AlaAsn Asp Ile Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ala

290 295 300 290 295 300

Asn Pro Gln Pro Lys Asp Asn Gly Leu Pro Leu Asp Lys Leu Leu LysAsn Pro Gln Pro Lys Asp Asn Gly Leu Pro Leu Asp Lys Leu Leu Lys

305 310 315 320305 310 315 320

Ser Ile Ile Gly Lys Glu Pro Val Leu Ile Pro Ile Ala Gly His HisSer Ile Ile Gly Lys Glu Pro Val Leu Ile Pro Ile Ala Gly His His

325 330 335 325 330 335

Ala Thr Glu Ile Glu Val Ala Arg Glu Thr His Phe Asp Gly Thr AsnAla Thr Glu Ile Glu Val Ala Arg Glu Thr His Phe Asp Gly Thr Asn

340 345 350 340 345 350

Tyr Leu Ala Ile Arg Pro Gly Val Val Ile Gly Tyr Ala Arg Asn GluTyr Leu Ala Ile Arg Pro Gly Val Val Ile Gly Tyr Ala Arg Asn Glu

355 360 365 355 360 365

Lys Thr Asn Glu Ala Leu Lys Asp Ala Gly Ile Thr Val Leu Pro PheLys Thr Asn Glu Ala Leu Lys Asp Ala Gly Ile Thr Val Leu Pro Phe

370 375 380 370 375 380

Lys Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met SerLys Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser

385 390 395 400385 390 395 400

Met Pro Leu Ser Arg Lys Asp Val Lys TrpMet Pro Leu Ser Arg Lys Asp Val Lys Trp

405 410 405 410

<210> 32<210> 32

<211> 411<211> 411

<212> PRT<212> PRT

<213> 泡沫支原体（Mycoplasma spumans）<213> Mycoplasma spamans

<400> 32<400> 32

Met Ser Val Phe Asp Ser Lys Phe Lys Gly Ile His Val Tyr Ser GluMet Ser Val Phe Asp Ser Lys Phe Lys Gly Ile His Val Tyr Ser Glu

1 5 10 151 5 10 15

Ile Gly Glu Leu Glu Ser Val Leu Val His Glu Pro Gly Arg Glu IleIle Gly Glu Leu Glu Ser Val Leu Val His Glu Pro Gly Arg Glu Ile

20 25 30 20 25 30

Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala IleAsp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile

35 40 45 35 40 45

Leu Glu Ser His Asp Ala Arg Lys Glu His Lys Gly Phe Val Ala GluLeu Glu Ser His Asp Ala Arg Lys Glu His Lys Gly Phe Val Ala Glu

50 55 60 50 55 60

Leu Lys Lys Gln Asn Val Asn Val Ile Glu Leu Thr Asp Leu Val AlaLeu Lys Lys Gln Asn Val Asn Val Ile Glu Leu Thr Asp Leu Val Ala

65 70 75 8065 70 75 80

Glu Thr Tyr Glu Leu Ala Ser Lys Glu Ala Gln Ala Lys Leu Ile GluGlu Thr Tyr Glu Leu Ala Ser Lys Glu Ala Gln Ala Lys Leu Ile Glu

85 90 95 85 90 95

Asp Phe Ile Glu Asp Ser Glu Pro Val Leu Asn Ala Glu Glu Ala GlnAsp Phe Ile Glu Asp Ser Glu Pro Val Leu Asn Ala Glu Glu Ala Gln

100 105 110 100 105 110

Ala Val Arg Lys Phe Leu Ser Glu Arg Lys Ser Thr Arg Glu Met ValAla Val Arg Lys Phe Leu Ser Glu Arg Lys Ser Thr Arg Glu Met Val

115 120 125 115 120 125

Glu Tyr Met Met Ser Gly Leu Thr Lys Tyr Glu Leu Gly Leu Glu SerGlu Tyr Met Met Ser Gly Leu Thr Lys Tyr Glu Leu Gly Leu Glu Ser

130 135 140 130 135 140

Ala Asp Arg Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe ThrAla Asp Arg Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr

145 150 155 160145 150 155 160

Arg Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr MetArg Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met

165 170 175 165 170 175

Lys Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ala Lys Phe Val PheLys Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ala Lys Phe Val Phe

180 185 190 180 185 190

Ser Asn His Pro Lys Leu Val Asn Thr Pro Arg Tyr Tyr Asp Pro SerSer Asn His Pro Lys Leu Val Asn Thr Pro Arg Tyr Tyr Asp Pro Ser

195 200 205 195 200 205

Met Lys Leu Pro Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn GluMet Lys Leu Pro Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Glu

210 215 220 210 215 220

Thr Leu Val Val Gly Cys Ser Glu Arg Thr Glu Leu Glu Thr Ile ThrThr Leu Val Val Gly Cys Ser Glu Arg Thr Glu Leu Glu Thr Ile Thr

225 230 235 240225 230 235 240

Leu Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu Val Glu Phe Lys ArgLeu Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu Val Glu Phe Lys Arg

245 250 255 245 250 255

Ile Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu AspIle Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp

260 265 270 260 265 270

Thr Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro IleThr Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile

275 280 285 275 280 285

Ala Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly GlyAla Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly

290 295 300 290 295 300

Glu Glu Pro Gln Pro Val Glu Asn Gly Leu Pro Leu Glu Glu Leu LeuGlu Glu Pro Gln Pro Val Glu Asn Gly Leu Pro Leu Glu Glu Leu Leu

305 310 315 320305 310 315 320

Ala Ser Ile Ile Asn Lys Lys Pro Thr Leu Ile Pro Ile Ala Gly GluAla Ser Ile Ile Asn Lys Lys Pro Thr Leu Ile Pro Ile Ala Gly Glu

325 330 335 325 330 335

Gly Ala Thr His Ile Asp Val Glu Arg Glu Thr His Phe Asp Gly ThrGly Ala Thr His Ile Asp Val Glu Arg Glu Thr His Phe Asp Gly Thr

340 345 350 340 345 350

Asn Tyr Leu Ala Ile Ala Pro Ala Leu Ile Ile Gly Tyr Ser Arg AsnAsn Tyr Leu Ala Ile Ala Pro Ala Leu Ile Ile Gly Tyr Ser Arg Asn

355 360 365 355 360 365

Glu Lys Thr Asn Ala Ala Leu Glu Lys Ala Gly Ile Thr Val Leu ProGlu Lys Thr Asn Ala Ala Leu Glu Lys Ala Gly Ile Thr Val Leu Pro

370 375 380 370 375 380

Phe His Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys MetPhe His Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met

385 390 395 400385 390 395 400

Ser Met Pro Leu Ser Arg Lys Asp Val Lys TrpSer Met Pro Leu Ser Arg Lys Asp Val Lys Trp

405 410 405 410

<210> 33<210> 33

<211> 410<211> 410

<212> PRT<212> PRT

<213> 耳支原体（Mycoplasma auris）<213> Mycoplasma auris

<400> 33<400> 33

Met Ser Val Phe Asp Ser Lys Phe Lys Gly Ile His Val Tyr Ser GluMet Ser Val Phe Asp Ser Lys Phe Lys Gly Ile His Val Tyr Ser Glu

1 5 10 151 5 10 15

Ile Gly Glu Leu Glu Thr Val Leu Val His Glu Pro Gly Arg Glu IleIle Gly Glu Leu Glu Thr Val Leu Val His Glu Pro Gly Arg Glu Ile

20 25 30 20 25 30

Asp Tyr Ile Thr Pro Lys Arg Leu Asp Glu Leu Leu Phe Ser Ala IleAsp Tyr Ile Thr Pro Lys Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile

35 40 45 35 40 45

Leu Glu Ser His Glu Ala Arg Lys Glu His Lys Gln Phe Val Ala GluLeu Glu Ser His Glu Ala Arg Lys Glu His Lys Gln Phe Val Ala Glu

50 55 60 50 55 60

Leu Lys Ala Asn Asp Ile Asn Val Val Glu Leu Thr Asp Leu Val AlaLeu Lys Ala Asn Asp Ile Asn Val Val Glu Leu Thr Asp Leu Val Ala

65 70 75 8065 70 75 80

Glu Thr Tyr Asp Leu Val Ser Gln Glu Leu Lys Asp Lys Leu Ile GluGlu Thr Tyr Asp Leu Val Ser Gln Glu Leu Lys Asp Lys Leu Ile Glu

85 90 95 85 90 95

Glu Phe Leu Asp Asp Ser Tyr Pro Val Leu Thr Glu Glu His Lys LysGlu Phe Leu Asp Asp Ser Tyr Pro Val Leu Thr Glu Glu His Lys Lys

100 105 110 100 105 110

Ala Val Arg Ser Phe Leu Lys Ser Arg Ser Ser Thr Arg Glu Leu IleAla Val Arg Ser Phe Leu Lys Ser Arg Ser Ser Thr Arg Glu Leu Ile

115 120 125 115 120 125

Glu Tyr Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Glu AlaGlu Tyr Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Glu Ala

130 135 140 130 135 140

Glu Gly Asp Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr ArgGlu Gly Asp Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg

145 150 155 160145 150 155 160

Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met ArgAsp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg

165 170 175 165 170 175

Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Ile Phe AspTyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Ile Phe Asp

180 185 190 180 185 190

Asn His Pro Lys Leu Val Asn Thr Pro Arg Tyr Tyr Asp Pro Ser LeuAsn His Pro Lys Leu Val Asn Thr Pro Arg Tyr Tyr Asp Pro Ser Leu

195 200 205 195 200 205

Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp ThrLys Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr

210 215 220 210 215 220

Leu Val Met Gly Val Ser Glu Arg Thr Asp Leu Glu Thr Val Thr LeuLeu Val Met Gly Val Ser Glu Arg Thr Asp Leu Glu Thr Val Thr Leu

225 230 235 240225 230 235 240

Leu Ala Lys Asn Ile Val Ala Asn Lys Glu Cys Glu Phe Lys Arg IleLeu Ala Lys Asn Ile Val Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile

245 250 255 245 250 255

Val Ala Ile Asn Val Pro His Trp Thr Asn Leu Met His Leu Asp ThrVal Ala Ile Asn Val Pro His Trp Thr Asn Leu Met His Leu Asp Thr

260 265 270 260 265 270

Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile AlaTrp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala

275 280 285 275 280 285

Asn Asp Tyr Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly AlaAsn Asp Tyr Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ala

290 295 300 290 295 300

Glu Pro Gln Pro Val Val Asn Glu Leu Pro Leu Asp Lys Leu Leu GluGlu Pro Gln Pro Val Val Asn Glu Leu Pro Leu Asp Lys Leu Leu Glu

305 310 315 320305 310 315 320

Ser Ile Ile His Lys Lys Pro Ile Leu Ile Pro Ile Ala Gly Glu GlySer Ile Ile His Lys Lys Pro Ile Leu Ile Pro Ile Ala Gly Glu Gly

325 330 335 325 330 335

Ala Ser Gln Ile Asp Leu Glu Arg Glu Thr His Phe Asp Gly Thr AsnAla Ser Gln Ile Asp Leu Glu Arg Glu Thr His Phe Asp Gly Thr Asn

340 345 350 340 345 350

Tyr Leu Val Leu Arg Pro Gly Val Val Val Gly Tyr Ala Arg Asn GluTyr Leu Val Leu Arg Pro Gly Val Val Val Gly Tyr Ala Arg Asn Glu

355 360 365 355 360 365

Lys Thr Asn Ala Ala Leu Glu Ala Val Gly Ile Lys Val Leu Pro PheLys Thr Asn Ala Ala Leu Glu Ala Val Gly Ile Lys Val Leu Pro Phe

370 375 380 370 375 380

Tyr Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ser Arg Cys Met SerTyr Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ser Arg Cys Met Ser

385 390 395 400385 390 395 400

Met Pro Leu Ser Arg Lys Asp Val Lys TrpMet Pro Leu Ser Arg Lys Asp Val Lys Trp

405 410 405 410

<210> 34<210> 34

<211> 410<211> 410

<212> PRT<212> PRT

<213> 猪滑液支原体（Mycoplasma hyosynoviae）<213> Mycoplasma hyosynoviae

<400> 34<400> 34

Met Ser Val Phe Asn Ser Lys Phe Lys Gly Ile His Val Tyr Ser GluMet Ser Val Phe Asn Ser Lys Phe Lys Gly Ile His Val Tyr Ser Glu

1 5 10 151 5 10 15

Ile Gly Asp Leu Glu Ser Val Leu Val His Glu Pro Gly Lys Glu IleIle Gly Asp Leu Glu Ser Val Leu Val His Glu Pro Gly Lys Glu Ile

20 25 30 20 25 30

Asp Tyr Ile Thr Pro Ser Arg Leu Asp Glu Leu Leu Phe Ser Ala IleAsp Tyr Ile Thr Pro Ser Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile

35 40 45 35 40 45

Leu Glu Ser Asn Asp Ala Arg Lys Glu His Lys Glu Phe Val Glu IleLeu Glu Ser Asn Asp Ala Arg Lys Glu His Lys Glu Phe Val Glu Ile

50 55 60 50 55 60

Leu Lys Lys Glu Gly Val Asn Val Val Glu Leu Val Asp Leu Ile AlaLeu Lys Lys Glu Gly Val Asn Val Val Glu Leu Val Asp Leu Ile Ala

65 70 75 8065 70 75 80

Glu Thr Ile Asp Leu Val Asp Ala Lys Lys Lys Glu Ala Leu Ile AspGlu Thr Ile Asp Leu Val Asp Ala Lys Lys Lys Glu Ala Leu Ile Asp

85 90 95 85 90 95

Glu Tyr Ile Glu Asp Ser Glu Pro Val Val Asp Ala Lys Val Lys ProGlu Tyr Ile Glu Asp Ser Glu Pro Val Val Asp Ala Lys Val Lys Pro

100 105 110 100 105 110

Leu Val Lys Lys Leu Leu Leu Gly Ile Lys Asp Thr Lys Glu Leu ValLeu Val Lys Lys Leu Leu Leu Gly Ile Lys Asp Thr Lys Glu Leu Val

115 120 125 115 120 125

Lys Leu Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Glu Ile Glu SerLys Leu Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Glu Ile Glu Ser

130 135 140 130 135 140

Glu Lys Glu Leu Ile Ile Asp Pro Met Pro Asn Leu Tyr Phe Thr ArgGlu Lys Glu Leu Ile Ile Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg

145 150 155 160145 150 155 160

Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met ArgAsp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg

165 170 175 165 170 175

Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Val Phe ArgTyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Val Phe Arg

180 185 190 180 185 190

Asn His Pro Lys Leu Thr Ser Thr Pro Trp Tyr Tyr Asp Pro Ala MetAsn His Pro Lys Leu Thr Ser Thr Pro Trp Tyr Tyr Asp Pro Ala Met

195 200 205 195 200 205

Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp ThrLys Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr

210 215 220 210 215 220

Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Asp Thr Ile Thr LeuLeu Val Val Gly Val Ser Glu Arg Thr Asp Leu Asp Thr Ile Thr Leu

225 230 235 240225 230 235 240

Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu Cys Glu Phe Lys Arg IleLeu Ala Lys Asn Ile Lys Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile

245 250 255 245 250 255

Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp ThrVal Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr

260 265 270 260 265 270

Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile AlaTrp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala

275 280 285 275 280 285

Asn Asp Ile Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly SerAsn Asp Ile Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ser

290 295 300 290 295 300

Glu Pro Gln Pro Lys Asp Asn Gly Leu Pro Leu Glu Lys Leu Leu GluGlu Pro Gln Pro Lys Asp Asn Gly Leu Pro Leu Glu Lys Leu Leu Glu

305 310 315 320305 310 315 320

Ser Ile Ile Gly Lys Lys Pro Val Leu Ile Pro Ile Ala Gly Cys CysSer Ile Ile Gly Lys Lys Pro Val Leu Ile Pro Ile Ala Gly Cys Cys

325 330 335 325 330 335

Ala Ser Asp Ile Glu Ile Ala Arg Glu Thr His Phe Asp Gly Thr AsnAla Ser Asp Ile Glu Ile Ala Arg Glu Thr His Phe Asp Gly Thr Asn

340 345 350 340 345 350

Tyr Leu Ala Ile Lys Pro Gly Val Val Ile Gly Tyr Ala Arg Asn GluTyr Leu Ala Ile Lys Pro Gly Val Val Ile Gly Tyr Ala Arg Asn Glu

355 360 365 355 360 365

Lys Thr Asn Lys Ala Leu Glu Lys Ala Gly Ile Lys Val Leu Pro PheLys Thr Asn Lys Ala Leu Glu Lys Ala Gly Ile Lys Val Leu Pro Phe

370 375 380 370 375 380

Lys Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met SerLys Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser

385 390 395 400385 390 395 400

Met Pro Leu Ser Arg Lys Asp Val Lys TrpMet Pro Leu Ser Arg Lys Asp Val Lys Trp

405 410 405 410

<210> 35<210> 35

<211> 409<211> 409

<212> PRT<212> PRT

<213> 泄殖腔支原体（Mycoplasma cloacale）<213> Mycoplasma cloacale

<400> 35<400> 35

Met Ser Val Phe Asp Lys Arg Phe Lys Gly Ile His Val Tyr Ser GluMet Ser Val Phe Asp Lys Arg Phe Lys Gly Ile His Val Tyr Ser Glu

1 5 10 151 5 10 15

Ile Gly Glu Leu Gln Thr Val Leu Val His Glu Pro Gly Arg Glu IleIle Gly Glu Leu Gln Thr Val Leu Val His Glu Pro Gly Arg Glu Ile

20 25 30 20 25 30

Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala IleAsp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile

35 40 45 35 40 45

Leu Glu Ser His Asp Ala Arg Lys Glu His Lys Glu Phe Val Lys IleLeu Glu Ser His Asp Ala Arg Lys Glu His Lys Glu Phe Val Lys Ile

50 55 60 50 55 60

Leu Glu Ser Gln Gly Ile Asn Val Val Glu Leu Thr Asp Leu Ile AlaLeu Glu Ser Gln Gly Ile Asn Val Val Glu Leu Thr Asp Leu Ile Ala

65 70 75 8065 70 75 80

Glu Thr Tyr Glu Leu Ala Ser Glu Glu Ala Lys Asp Asn Leu Ile GluGlu Thr Tyr Glu Leu Ala Ser Glu Glu Ala Lys Asp Asn Leu Ile Glu

85 90 95 85 90 95

Glu Phe Leu Asp Glu Ser Glu Pro Val Leu Ser Glu Glu His Arg IleGlu Phe Leu Asp Glu Ser Glu Pro Val Leu Ser Glu Glu His Arg Ile

100 105 110 100 105 110

Leu Val Arg Asn Phe Leu Lys Gly Ile Thr Lys Thr Lys Glu Leu ValLeu Val Arg Asn Phe Leu Lys Gly Ile Thr Lys Thr Lys Glu Leu Val

115 120 125 115 120 125

Lys Met Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Glu AlaLys Met Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Glu Ala

130 135 140 130 135 140

Asp Arg Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr ArgAsp Arg Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg

145 150 155 160145 150 155 160

Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met ArgAsp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg

165 170 175 165 170 175

Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Ile Phe GluTyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Ile Phe Glu

180 185 190 180 185 190

Asn His Pro Lys Leu Val Ser Thr Pro Ile Tyr Tyr His Pro Ser GlnAsn His Pro Lys Leu Val Ser Thr Pro Ile Tyr Tyr His Pro Ser Gln

195 200 205 195 200 205

Gly Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp ThrGly Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr

210 215 220 210 215 220

Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Gln Thr Ile Thr LeuLeu Val Val Gly Val Ser Glu Arg Thr Asp Leu Gln Thr Ile Thr Leu

225 230 235 240225 230 235 240

Leu Ala Lys Asn Ile Lys Ala Asn Glu Glu Cys Glu Phe Lys Arg IleLeu Ala Lys Asn Ile Lys Ala Asn Glu Glu Cys Glu Phe Lys Arg Ile

245 250 255 245 250 255

Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp ThrVal Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr

260 265 270 260 265 270

Trp Leu Thr Met Leu Asp Lys Asn Lys Phe Leu Tyr Ser Pro Ile AlaTrp Leu Thr Met Leu Asp Lys Asn Lys Phe Leu Tyr Ser Pro Ile Ala

275 280 285 275 280 285

Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly AspAsn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Asp

290 295 300 290 295 300

Glu Pro Gln Pro Val Asp Asn Gly Leu Pro Leu Asn Glu Leu Leu AlaGlu Pro Gln Pro Val Asp Asn Gly Leu Pro Leu Asn Glu Leu Leu Ala

305 310 315 320305 310 315 320

Ser Ile Ile Gly Glu Glu Pro Val Leu Val Pro Ile Ala Gly Glu GlySer Ile Ile Gly Glu Glu Pro Val Leu Val Pro Ile Ala Gly Glu Gly

325 330 335 325 330 335

Ala Ser Lys Met Asp Ile Glu Arg Glu Thr His Phe Asp Gly Thr AsnAla Ser Lys Met Asp Ile Glu Arg Glu Thr His Phe Asp Gly Thr Asn

340 345 350 340 345 350

Tyr Leu Ala Ile Ala Pro Gly Val Val Val Gly Tyr Ser Arg Asn GluTyr Leu Ala Ile Ala Pro Gly Val Val Val Gly Tyr Ser Arg Asn Glu

355 360 365 355 360 365

Lys Thr Asn Ala Ala Leu Glu Lys Ala Gly Ile Lys Val Leu Pro PheLys Thr Asn Ala Ala Leu Glu Lys Ala Gly Ile Lys Val Leu Pro Phe

370 375 380 370 375 380

Lys Gly His Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met SerLys Gly His Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser

385 390 395 400385 390 395 400

Met Pro Leu Tyr Arg Lys Asp Val LysMet Pro Leu Tyr Arg Lys Asp Val Lys

405 405

<210> 36<210> 36

<211> 412<211> 412

<212> PRT<212> PRT

<213> 产碱支原体（Mycoplasma alkalescens）<213> Mycoplasma alkalescens

<400> 36<400> 36

Met Ser Val Phe Asp Ser Lys Phe Lys Gly Ile His Val Tyr Ser GluMet Ser Val Phe Asp Ser Lys Phe Lys Gly Ile His Val Tyr Ser Glu

1 5 10 151 5 10 15

Ile Gly Glu Leu Glu Ser Val Leu Val His Glu Pro Gly His Glu IleIle Gly Glu Leu Glu Ser Val Leu Val His Glu Pro Gly His Glu Ile

20 25 30 20 25 30

Asp Tyr Ile Thr Pro Ser Arg Leu Asp Glu Leu Leu Phe Ser Ala MetAsp Tyr Ile Thr Pro Ser Arg Leu Asp Glu Leu Leu Phe Ser Ala Met

35 40 45 35 40 45

Leu Glu Ser His Asp Ala Arg Lys Glu His Lys Gln Phe Val Ala GluLeu Glu Ser His Asp Ala Arg Lys Glu His Lys Gln Phe Val Ala Glu

50 55 60 50 55 60

Leu Lys Ala Asn Asn Val Asn Val Ile Glu Leu Thr Asp Leu Val AlaLeu Lys Ala Asn Asn Val Asn Val Ile Glu Leu Thr Asp Leu Val Ala

65 70 75 8065 70 75 80

Glu Thr Tyr Asp Leu Ala Ser Gln Glu Ala Lys Asp Lys Leu Ile GluGlu Thr Tyr Asp Leu Ala Ser Gln Glu Ala Lys Asp Lys Leu Ile Glu

85 90 95 85 90 95

Glu Phe Leu Glu Asp Ser Glu Pro Val Leu Ser Glu Glu Asn Lys IleGlu Phe Leu Glu Asp Ser Glu Pro Val Leu Ser Glu Glu Asn Lys Ile

100 105 110 100 105 110

Ala Val Arg Asp Phe Leu Lys Ser Arg Lys Thr Thr Arg Glu Leu IleAla Val Arg Asp Phe Leu Lys Ser Arg Lys Thr Thr Arg Glu Leu Ile

115 120 125 115 120 125

Glu Val Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Lys AsnGlu Val Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Lys Asn

130 135 140 130 135 140

Cys Lys Cys Gln Asp Leu Val Val Asp Pro Met Pro Asn Leu Tyr PheCys Lys Cys Gln Asp Leu Val Val Asp Pro Met Pro Asn Leu Tyr Phe

145 150 155 160145 150 155 160

Thr Arg Asp Pro Phe Ala Ser Val Gly Asn Gly Ile Thr Ile His TyrThr Arg Asp Pro Phe Ala Ser Val Gly Asn Gly Ile Thr Ile His Tyr

165 170 175 165 170 175

Met Arg Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe IleMet Arg Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Ile

180 185 190 180 185 190

Phe Ala Asn His Pro Lys Leu Val Asn Thr Pro Ile Tyr Tyr His ProPhe Ala Asn His Pro Lys Leu Val Asn Thr Pro Ile Tyr Tyr His Pro

195 200 205 195 200 205

Ser Leu Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn AsnSer Leu Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn

210 215 220 210 215 220

Asp Thr Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Glu Thr IleAsp Thr Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Glu Thr Ile

225 230 235 240225 230 235 240

Thr Leu Leu Ala Lys Asn Ile Val Ala Asn Lys Glu Cys Glu Phe LysThr Leu Leu Ala Lys Asn Ile Val Ala Asn Lys Glu Cys Glu Phe Lys

245 250 255 245 250 255

Arg Ile Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His LeuArg Ile Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu

260 265 270 260 265 270

Asp Thr Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser ProAsp Thr Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro

275 280 285 275 280 285

Ile Ala Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn GlyIle Ala Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly

290 295 300 290 295 300

Gly Ala Glu Pro Lys Pro Val Glu Asn Gly Ser Ser Leu Glu Ala IleGly Ala Glu Pro Lys Pro Val Glu Asn Gly Ser Ser Leu Glu Ala Ile

305 310 315 320305 310 315 320

Leu Glu Ser Ile Ile His Lys Lys Pro Ile Leu Ile Pro Ile Gly GlyLeu Glu Ser Ile Ile His Lys Lys Pro Ile Leu Ile Pro Ile Gly Gly

325 330 335 325 330 335

Asp Ser Ala Ser Gln Ile Glu Val Glu Arg Glu Thr His Phe Asp GlyAsp Ser Ala Ser Gln Ile Glu Val Glu Arg Glu Thr His Phe Asp Gly

340 345 350 340 345 350

Thr Asn Tyr Leu Ala Ile Arg Pro Gly Val Val Ile Gly Tyr Ser ArgThr Asn Tyr Leu Ala Ile Arg Pro Gly Val Val Ile Gly Tyr Ser Arg

355 360 365 355 360 365

Asn Val Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Lys Val IleAsn Val Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Lys Val Ile

370 375 380 370 375 380

Pro Phe His Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg CysPro Phe His Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys

385 390 395 400385 390 395 400

Met Ser Met Pro Leu Ser Arg Lys Asp Val Lys TrpMet Ser Met Pro Leu Ser Arg Lys Asp Val Lys Trp

405 410 405 410

<210> 37<210> 37

<211> 401<211> 401

<212> PRT<212> PRT

<213> 惰性支原体（Mycoplasma iners）<213> Mycoplasma iners

<400> 37<400> 37

Met Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Val Leu Lys Glu ValMet Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Val Leu Lys Glu Val

1 5 10 151 5 10 15

Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ala Pro Ser ArgLeu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ala Pro Ser Arg

20 25 30 20 25 30

Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Ser Ala Ala IleLeu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Ser Ala Ala Ile

35 40 45 35 40 45

Gln Glu His Lys Ser Phe Leu Lys Ile Leu Gln Asp Arg Gly Ile LysGln Glu His Lys Ser Phe Leu Lys Ile Leu Gln Asp Arg Gly Ile Lys

50 55 60 50 55 60

Thr Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Lys His Tyr AlaThr Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Lys His Tyr Ala

65 70 75 8065 70 75 80

Ser Glu Ala Glu Lys Glu Ala Phe Ile Glu Lys Tyr Leu Asp Glu AlaSer Glu Ala Glu Lys Glu Ala Phe Ile Glu Lys Tyr Leu Asp Glu Ala

85 90 95 85 90 95

Thr Pro Val Leu Ser Lys Asp Met Arg Ala Lys Val Lys Asn Tyr IleThr Pro Val Leu Ser Lys Asp Met Arg Ala Lys Val Lys Asn Tyr Ile

100 105 110 100 105 110

Leu Ser Met Gln Gly Glu Pro Val Lys Met Val Arg Thr Met Met AlaLeu Ser Met Gln Gly Glu Pro Val Lys Met Val Arg Thr Met Met Ala

115 120 125 115 120 125

Gly Val Ser Lys Gln Glu Leu Asn Val Glu Ser Glu Val Glu Leu IleGly Val Ser Lys Gln Glu Leu Asn Val Glu Ser Glu Val Glu Leu Ile

130 135 140 130 135 140

Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala SerVal Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser

145 150 155 160145 150 155 160

Ala Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg LysAla Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg Lys

165 170 175 165 170 175

Arg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ser Ile His Pro Glu TyrArg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ser Ile His Pro Glu Tyr

180 185 190 180 185 190

Lys Lys Thr Pro His Trp Phe Asp Arg Leu Asp Asn Gly Ser Ile GluLys Lys Thr Pro His Trp Phe Asp Arg Leu Asp Asn Gly Ser Ile Glu

195 200 205 195 200 205

Gly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly ValGly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly Val

210 215 220 210 215 220

Ser Glu Arg Thr Asn Lys Glu Ala Ile Ile Thr Ile Ala Lys His IleSer Glu Arg Thr Asn Lys Glu Ala Ile Ile Thr Ile Ala Lys His Ile

225 230 235 240225 230 235 240

Gln Asp Asn Lys Glu Ala Gln Phe Lys Lys Ile Val Ala Ile Asn ValGln Asp Asn Lys Glu Ala Gln Phe Lys Lys Ile Val Ala Ile Asn Val

245 250 255 245 250 255

Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met ValPro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Val

260 265 270 260 265 270

Asp Lys Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu LysAsp Lys Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu Lys

275 280 285 275 280 285

Val Trp Glu Ile Asp Leu Ser Lys Pro Ile Glu Met Val Glu Thr AsnVal Trp Glu Ile Asp Leu Ser Lys Pro Ile Glu Met Val Glu Thr Asn

290 295 300 290 295 300

Lys Pro Leu Ala Glu Val Leu Glu Ser Ile Ile Gly Glu Lys Pro IleLys Pro Leu Ala Glu Val Leu Glu Ser Ile Ile Gly Glu Lys Pro Ile

305 310 315 320305 310 315 320

Leu Ile Pro Ile Ala Gly Lys Asp Ala Thr Gln Leu Asp Ile Asp IleLeu Ile Pro Ile Ala Gly Lys Asp Ala Thr Gln Leu Asp Ile Asp Ile

325 330 335 325 330 335

Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly ValGlu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly Val

340 345 350 340 345 350

Val Val Gly Tyr Ser Arg Asn Val Lys Thr Glu Ala Ala Leu Arg AlaVal Val Gly Tyr Ser Arg Asn Val Lys Thr Glu Ala Ala Leu Arg Ala

355 360 365 355 360 365

Ala Gly Val Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu GlyAla Gly Val Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu Gly

370 375 380 370 375 380

Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp ValMet Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp Val

385 390 395 400385 390 395 400

LysLys

<210> 38<210> 38

<211> 401<211> 401

<212> PRT<212> PRT

<213> 鸡支原体（Mycoplasma gallinarum）<213> Mycoplasma gallinarum

<400> 38<400> 38

Met Ser Lys Ile Arg Val Tyr Ser Glu Ile Gly Asn Leu Lys Lys ValMet Ser Lys Ile Arg Val Tyr Ser Glu Ile Gly Asn Leu Lys Lys Val

1 5 10 151 5 10 15

Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser ArgLeu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser Arg

20 25 30 20 25 30

Leu Glu Glu Leu Leu Phe Ser Ala Val Leu Glu Pro Asn Ala Ala IleLeu Glu Glu Leu Leu Phe Ser Ala Val Leu Glu Pro Asn Ala Ala Ile

35 40 45 35 40 45

Glu Glu His Lys Arg Phe Val Lys Leu Leu Glu Asp Arg Gly Ile GlnGlu Glu His Lys Arg Phe Val Lys Leu Leu Glu Asp Arg Gly Ile Gln

50 55 60 50 55 60

Ala Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Val Lys Tyr AlaAla Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Val Lys Tyr Ala

65 70 75 8065 70 75 80

Thr Ala Glu Gln Lys Ala Ala Phe Ile Glu Lys Tyr Leu Asp Glu AlaThr Ala Glu Gln Lys Ala Ala Phe Ile Glu Lys Tyr Leu Asp Glu Ala

85 90 95 85 90 95

Thr Pro Ala Leu Ser Ala Glu Asn Arg Glu Arg Ala Lys Lys Tyr IleThr Pro Ala Leu Ser Ala Glu Asn Arg Glu Arg Ala Lys Lys Tyr Ile

100 105 110 100 105 110

Leu Ser Leu Glu Met Gln Pro Val Lys Met Ile Arg Thr Met Met AlaLeu Ser Leu Glu Met Gln Pro Val Lys Met Ile Arg Thr Met Met Ala

115 120 125 115 120 125

Gly Leu Ser Lys Tyr Glu Leu Asn Val Glu Ser Asn Ile Glu Leu IleGly Leu Ser Lys Tyr Glu Leu Asn Val Glu Ser Asn Ile Glu Leu Ile

130 135 140 130 135 140

Ile Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala SerIle Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser

145 150 155 160145 150 155 160

Ala Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg LysAla Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg Lys

165 170 175 165 170 175

Arg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ala Ile His Pro Glu TyrArg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ala Ile His Pro Glu Tyr

180 185 190 180 185 190

Lys Glu Thr Pro His Trp Phe Asp Arg Leu Asp His Gly Ser Ile GluLys Glu Thr Pro His Trp Phe Asp Arg Leu Asp His Gly Ser Ile Glu

195 200 205 195 200 205

Gly Gly Asp Val Phe Val Tyr Asn Lys Asp Thr Leu Val Ile Gly ValGly Gly Asp Val Phe Val Tyr Asn Lys Asp Thr Leu Val Ile Gly Val

210 215 220 210 215 220

Ser Glu Arg Thr Asn Lys Glu Ala Ile Ile Thr Ile Ala Lys His IleSer Glu Arg Thr Asn Lys Glu Ala Ile Ile Thr Ile Ala Lys His Ile

225 230 235 240225 230 235 240

Gln Asp Asn Lys Glu Ala Glu Phe Lys Lys Ile Val Ala Ile Asn ValGln Asp Asn Lys Glu Ala Glu Phe Lys Lys Ile Val Ala Ile Asn Val

245 250 255 245 250 255

Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met ValPro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Val

260 265 270 260 265 270

Asp Lys Asn Lys Phe Ile Tyr Ser Pro Asn Met Leu Ser Val Leu LysAsp Lys Asn Lys Phe Ile Tyr Ser Pro Asn Met Leu Ser Val Leu Lys

275 280 285 275 280 285

Ile Trp Glu Ile Asp Leu Ala Lys Pro Ile Glu Met Val Glu Ser AsnIle Trp Glu Ile Asp Leu Ala Lys Pro Ile Glu Met Val Glu Ser Asn

290 295 300 290 295 300

Lys Ser Leu Thr Glu Val Leu Glu Ser Ile Ile Gly Glu Lys Pro IleLys Ser Leu Thr Glu Val Leu Glu Ser Ile Ile Gly Glu Lys Pro Ile

305 310 315 320305 310 315 320

Leu Ile Pro Ile Ala Gly Glu Gly Ala Ser Gln Leu Asp Ile Asp IleLeu Ile Pro Ile Ala Gly Glu Gly Ala Ser Gln Leu Asp Ile Asp Ile

325 330 335 325 330 335

Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly ValGlu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly Val

340 345 350 340 345 350

Val Val Gly Tyr Ser Arg Asn Glu Lys Thr Glu Lys Ala Leu Lys AlaVal Val Gly Tyr Ser Arg Asn Glu Lys Thr Glu Lys Ala Leu Lys Ala

355 360 365 355 360 365

Ala Gly Ile Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu GlyAla Gly Ile Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu Gly

370 375 380 370 375 380

Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp ValMet Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp Val

385 390 395 400385 390 395 400

LysLys

<210> 39<210> 39

<211> 405<211> 405

<212> PRT<212> PRT

<213> 梨支原体（Mycoplasma pirum）<213> Mycoplasma pirum

<400> 39<400> 39

Met Asn Ser Asn Gln Lys Gly Ile His Val Tyr Ser Glu Ile Gly LysMet Asn Ser Asn Gln Lys Gly Ile His Val Tyr Ser Glu Ile Gly Lys

1 5 10 151 5 10 15

Leu Lys Glu Val Leu Val His Arg Pro Gly Arg Glu Leu Asp Phe LeuLeu Lys Glu Val Leu Val His Arg Pro Gly Arg Glu Leu Asp Phe Leu

20 25 30 20 25 30

Asp Pro Thr Arg Leu Asp Glu Leu Leu Phe Ala Ala Thr Leu Glu AlaAsp Pro Thr Arg Leu Asp Glu Leu Leu Phe Ala Ala Thr Leu Glu Ala

35 40 45 35 40 45

Glu Thr Ala Arg Leu Glu His Asp Asn Phe Thr Asn Ala Leu Lys AsnGlu Thr Ala Arg Leu Glu His Asp Asn Phe Thr Asn Ala Leu Lys Asn

50 55 60 50 55 60

Gln Gly Val Thr Val Ile Glu Leu Ala Asp Leu Val Ala Gln Thr TyrGln Gly Val Thr Val Ile Glu Leu Ala Asp Leu Val Ala Gln Thr Tyr

65 70 75 8065 70 75 80

Ser Ser Ser Thr Pro Thr Ile Lys Ala Ala Phe Ile Asn Lys Tyr LeuSer Ser Ser Thr Pro Thr Ile Lys Ala Ala Phe Ile Asn Lys Tyr Leu

85 90 95 85 90 95

Asp Glu Ala Thr Pro Ala Leu Thr Thr Lys Leu Arg Thr Leu Val LysAsp Glu Ala Thr Pro Ala Leu Thr Thr Lys Leu Arg Thr Leu Val Lys

100 105 110 100 105 110

Asp Phe Leu Thr Lys Gln Lys Ser Val Arg Lys Met Val Asp Tyr MetAsp Phe Leu Thr Lys Gln Lys Ser Val Arg Lys Met Val Asp Tyr Met

115 120 125 115 120 125

Ile Gly Gly Ile Leu Ser Thr Asp Leu Asn Ile Lys Gly Lys Pro GluIle Gly Gly Ile Leu Ser Thr Asp Leu Asn Ile Lys Gly Lys Pro Glu

130 135 140 130 135 140

Leu Ile Val Glu Pro Met Pro Asn Ala Tyr Phe Thr His Asp Pro PheLeu Ile Val Glu Pro Met Pro Asn Ala Tyr Phe Thr His Asp Pro Phe

145 150 155 160145 150 155 160

Ala Ser Val Gly Asn Gly Val Thr Leu His Tyr Met Lys His Asn ValAla Ser Val Gly Asn Gly Val Thr Leu His Tyr Met Lys His Asn Val

165 170 175 165 170 175

Arg Arg Arg Glu Val Leu Phe Ser Glu Phe Ile Phe Asn Asn Asn GluArg Arg Arg Glu Val Leu Phe Ser Glu Phe Ile Phe Asn Asn Asn Glu

180 185 190 180 185 190

Arg Phe Gln Asn Thr Pro Arg Tyr Ile Val Pro Thr Lys Gly Leu AspArg Phe Gln Asn Thr Pro Arg Tyr Ile Val Pro Thr Lys Gly Leu Asp

195 200 205 195 200 205

Ile Glu Gly Gly Asp Val Phe Val Tyr Asn Lys Asn Thr Leu Val ValIle Glu Gly Gly Asp Val Phe Val Tyr Asn Lys Asn Thr Leu Val Val

210 215 220 210 215 220

Gly Val Ser Glu Arg Thr Lys Met Val Thr Ile Lys Glu Leu Ala LysGly Val Ser Glu Arg Thr Lys Met Val Thr Ile Lys Glu Leu Ala Lys

225 230 235 240225 230 235 240

Asn Ile Leu Lys Asn Lys Glu Cys Leu Phe Lys Lys Ile Tyr Ala IleAsn Ile Leu Lys Asn Lys Glu Cys Leu Phe Lys Lys Ile Tyr Ala Ile

245 250 255 245 250 255

Asn Val Pro Lys Met Pro Asn Leu Met His Leu Asp Thr Trp Leu ThrAsn Val Pro Lys Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr

260 265 270 260 265 270

Met Leu Asp His Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser ValMet Leu Asp His Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val

275 280 285 275 280 285

Leu Lys Ile Trp Glu Ile Asp Ile Ser Ser Gly Lys Ser Ile Ser SerLeu Lys Ile Trp Glu Ile Asp Ile Ser Ser Gly Lys Ser Ile Ser Ser

290 295 300 290 295 300

Pro Lys Glu Leu Asn Met Asp Leu Ser Lys Ala Leu Ser Ile Ile IlePro Lys Glu Leu Asn Met Asp Leu Ser Lys Ala Leu Ser Ile Ile Ile

305 310 315 320305 310 315 320

Gly Lys Lys Pro Ile Leu Ile Pro Val Ala Gly Glu Asn Ala Ser GlnGly Lys Lys Pro Ile Leu Ile Pro Val Ala Gly Glu Asn Ala Ser Gln

325 330 335 325 330 335

Ile Asp Ile Asn Ile Glu Thr Asn Phe Asp Ala Thr Asn Tyr Leu ValIle Asp Ile Asn Ile Glu Thr Asn Phe Asp Ala Thr Asn Tyr Leu Val

340 345 350 340 345 350

Thr Gln Pro Gly Val Val Val Gly Tyr Ser Arg Asn Lys Lys Thr GluThr Gln Pro Gly Val Val Val Gly Tyr Ser Arg Asn Lys Lys Thr Glu

355 360 365 355 360 365

Ala Ala Leu Ile Lys Ala Gly Ile Glu Val Ile Pro Phe Gln Gly AsnAla Ala Leu Ile Lys Ala Gly Ile Glu Val Ile Pro Phe Gln Gly Asn

370 375 380 370 375 380

Gln Leu Ser Leu Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro LeuGln Leu Ser Leu Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu

385 390 395 400385 390 395 400

Ile Arg Glu Asp ValIle Arg Glu Asp Val

405 405

<210> 40<210> 40

<211> 404<211> 404

<212> PRT<212> PRT

<213> 灵长类支原体（Mycoplasma primatum）<213> Mycoplasma primatum

<400> 40<400> 40

Met Ser Lys Ser Lys Ile Asn Val Tyr Ser Glu Tyr Gly Asn Leu LysMet Ser Lys Ser Lys Ile Asn Val Tyr Ser Glu Tyr Gly Asn Leu Lys

1 5 10 151 5 10 15

Glu Val Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Thr ProGlu Val Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Thr Pro

20 25 30 20 25 30

Ser Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Lys SerSer Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Lys Ser

35 40 45 35 40 45

Ala Ile Ala Glu His Lys Ser Phe Cys Gln Ile Leu Lys Asp Asn LysAla Ile Ala Glu His Lys Ser Phe Cys Gln Ile Leu Lys Asp Asn Lys

50 55 60 50 55 60

Val Lys Ala Ile Gln Leu Asp Glu Leu Val Ala Ala Thr Tyr Lys GlyVal Lys Ala Ile Gln Leu Asp Glu Leu Val Ala Ala Thr Tyr Lys Gly

65 70 75 8065 70 75 80

Val Ser Glu Ser Val Gln Asn Ser Phe Val Glu Arg Trp Leu Asp GluVal Ser Glu Ser Val Gln Asn Ser Phe Val Glu Arg Trp Leu Asp Glu

85 90 95 85 90 95

Cys Glu Pro Lys Leu Glu Asn Asn Val Arg Pro Ile Val Lys Glu TyrCys Glu Pro Lys Leu Glu Asn Asn Val Arg Pro Ile Val Lys Glu Tyr

100 105 110 100 105 110

Leu Leu Lys Ala Ala Glu Gln Ser Val Lys Lys Met Ile Arg Ile MetLeu Leu Lys Ala Ala Glu Gln Ser Val Lys Lys Met Ile Arg Ile Met

115 120 125 115 120 125

Met Ala Gly Ile Asp Lys Arg Glu Ile Gly Val Glu Ser Glu Val AspMet Ala Gly Ile Asp Lys Arg Glu Ile Gly Val Glu Ser Glu Val Asp

130 135 140 130 135 140

Phe Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro PhePhe Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe

145 150 155 160145 150 155 160

Ala Ser Val Gly Asn Gly Ile Thr Leu His His Met Lys Tyr Val ValAla Ser Val Gly Asn Gly Ile Thr Leu His His Met Lys Tyr Val Val

165 170 175 165 170 175

Arg Gln Arg Glu Thr Leu Phe Ser Glu Phe Ile Phe Asp Asn His ProArg Gln Arg Glu Thr Leu Phe Ser Glu Phe Ile Phe Asp Asn His Pro

180 185 190 180 185 190

Asp Tyr Lys Phe Val Pro Arg Tyr Phe Asp Arg Asp Asp Glu Gly LysAsp Tyr Lys Phe Val Pro Arg Tyr Phe Asp Arg Asp Asp Glu Gly Lys

195 200 205 195 200 205

Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Ser Lys Thr Leu Val ValIle Glu Gly Gly Asp Val Phe Ile Tyr Asn Ser Lys Thr Leu Val Val

210 215 220 210 215 220

Gly Ile Ser Glu Arg Thr Asn Lys Asp Ala Ile Arg Ile Val Ala LysGly Ile Ser Glu Arg Thr Asn Lys Asp Ala Ile Arg Ile Val Ala Lys

225 230 235 240225 230 235 240

Lys Ile Gln Ala Asn Ala Asp Ala Lys Phe Glu Lys Ile Phe Ala IleLys Ile Gln Ala Asn Ala Asp Ala Lys Phe Glu Lys Ile Phe Ala Ile

245 250 255 245 250 255

Asn Val Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu ThrAsn Val Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr

260 265 270 260 265 270

Met Leu Asp Ser Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser ValMet Leu Asp Ser Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val

275 280 285 275 280 285

Leu Lys Val Trp Glu Ile Asn Leu Asp Asp Pro Ala Leu Glu Trp LysLeu Lys Val Trp Glu Ile Asn Leu Asp Asp Pro Ala Leu Glu Trp Lys

290 295 300 290 295 300

Glu Ile Ser Gly Ser Leu Glu Glu Ile Leu Thr Tyr Ile Ile Gly LysGlu Ile Ser Gly Ser Leu Glu Glu Ile Leu Thr Tyr Ile Ile Gly Lys

305 310 315 320305 310 315 320

Lys Pro Ile Leu Ile Pro Ile Ala Gly Lys Gly Ala Ser Gln Phe GluLys Pro Ile Leu Ile Pro Ile Ala Gly Lys Gly Ala Ser Gln Phe Glu

325 330 335 325 330 335

Ile Asp Ile Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Ala Ile AlaIle Asp Ile Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Ala Ile Ala

340 345 350 340 345 350

Pro Ser Val Val Ile Gly Tyr Ser Arg Asn Glu Leu Thr Glu Lys AlaPro Ser Val Val Ile Gly Tyr Ser Arg Asn Glu Leu Thr Glu Lys Ala

355 360 365 355 360 365

Leu Lys Lys Ala Gly Val Lys Val Leu Ser Leu Asp Gly Asn Gln LeuLeu Lys Lys Ala Gly Val Lys Val Leu Ser Leu Asp Gly Asn Gln Leu

370 375 380 370 375 380

Ser Leu Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Ile ArgSer Leu Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Ile Arg

385 390 395 400385 390 395 400

Glu Asp Val LysGlu Asp Val Lys

<210> 41<210> 41

<211> 401<211> 401

<212> PRT<212> PRT

<213> 产脂支原体（Mycoplasma lipofaciens）<213> Mycoplasma lipofaciens

<400> 41<400> 41

Met Ser Lys Ile Asn Val Tyr Ser Glu Val Gly Val Leu Lys Glu ValMet Ser Lys Ile Asn Val Tyr Ser Glu Val Gly Val Leu Lys Glu Val

1 5 10 151 5 10 15

Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Val Ala Pro Ser ArgLeu Val His Thr Pro Gly Asp Glu Ile Arg Arg Val Ala Pro Ser Arg

20 25 30 20 25 30

Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Gln Asp Ala IleLeu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Gln Asp Ala Ile

35 40 45 35 40 45

Ala Glu His Lys Arg Phe Ile Lys Ile Leu Glu Asp Asn Asn Ile LysAla Glu His Lys Arg Phe Ile Lys Ile Leu Glu Asp Asn Asn Ile Lys

50 55 60 50 55 60

Val Ile Gln Leu Asp Glu Leu Val Ser Glu Thr Trp Glu Lys Ala ThrVal Ile Gln Leu Asp Glu Leu Val Ser Glu Thr Trp Glu Lys Ala Thr

65 70 75 8065 70 75 80

Ala Glu Gln Arg Asp Ala Phe Ile Glu Lys Trp Leu Asp Glu Ala GluAla Glu Gln Arg Asp Ala Phe Ile Glu Lys Trp Leu Asp Glu Ala Glu

85 90 95 85 90 95

Pro Val Leu Asp Ala Lys Leu Arg Glu Thr Val Lys Lys Tyr Leu LeuPro Val Leu Asp Ala Lys Leu Arg Glu Thr Val Lys Lys Tyr Leu Leu

100 105 110 100 105 110

Ser Leu Asn Pro Val Lys Lys Met Val Arg Thr Met Met Ala Gly IleSer Leu Asn Pro Val Lys Lys Met Val Arg Thr Met Met Ala Gly Ile

115 120 125 115 120 125

Asp Lys Lys Glu Leu Lys Ile Glu Leu Asp Arg Asp Leu Val Val AspAsp Lys Lys Glu Leu Lys Ile Glu Leu Asp Arg Asp Leu Val Val Asp

130 135 140 130 135 140

Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser Ala GlyPro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser Ala Gly

145 150 155 160145 150 155 160

Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Thr Arg Lys Arg GluAsn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Thr Arg Lys Arg Glu

165 170 175 165 170 175

Thr Ile Phe Ala Glu Phe Ile Phe Asn Ile His Pro Asp Tyr Lys ThrThr Ile Phe Ala Glu Phe Ile Phe Asn Ile His Pro Asp Tyr Lys Thr

180 185 190 180 185 190

Thr Pro His Trp Phe Asp Arg Leu Asp Lys Gly Asn Ile Glu Gly GlyThr Pro His Trp Phe Asp Arg Leu Asp Lys Gly Asn Ile Glu Gly Gly

195 200 205 195 200 205

Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Leu Gly Val Ser GluAsp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Leu Gly Val Ser Glu

210 215 220 210 215 220

Arg Thr Asn Lys Asp Ala Val Met Thr Ile Ala Lys His Ile Gln SerArg Thr Asn Lys Asp Ala Val Met Thr Ile Ala Lys His Ile Gln Ser

225 230 235 240225 230 235 240

Asn Glu Gln Ala Lys Phe Lys Lys Leu Val Ala Ile Asn Val Pro ProAsn Glu Gln Ala Lys Phe Lys Lys Leu Val Ala Ile Asn Val Pro Pro

245 250 255 245 250 255

Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Val Asp HisMet Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Val Asp His

260 265 270 260 265 270

Asp Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu Lys Ile TrpAsp Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu Lys Ile Trp

275 280 285 275 280 285

Glu Ile Asp Leu Thr Pro Gly Lys Glu Ile Glu Met Val Glu Ser ThrGlu Ile Asp Leu Thr Pro Gly Lys Glu Ile Glu Met Val Glu Ser Thr

290 295 300 290 295 300

Lys Ser Leu Ser Asp Met Leu Glu Ser Ile Ile Gly Lys Lys Pro ValLys Ser Leu Ser Asp Met Leu Glu Ser Ile Ile Gly Lys Lys Pro Val

305 310 315 320305 310 315 320

Leu Ile Pro Ile Ala Gly Lys Asp Ala Ser Gln Leu Asp Ile Asp IleLeu Ile Pro Ile Ala Gly Lys Asp Ala Ser Gln Leu Asp Ile Asp Ile

325 330 335 325 330 335

Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Arg Pro Gly ValGlu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Arg Pro Gly Val

340 345 350 340 345 350

Val Val Gly Tyr Ser Arg Asn Cys Leu Thr Glu Gln Ala Leu Lys AspVal Val Gly Tyr Ser Arg Asn Cys Leu Thr Glu Gln Ala Leu Lys Asp

355 360 365 355 360 365

Ala Gly Val Thr Val Leu Ser Phe Asp Gly Asn Gln Leu Ser Leu GlyAla Gly Val Thr Val Leu Ser Phe Asp Gly Asn Gln Leu Ser Leu Gly

370 375 380 370 375 380

Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp IleMet Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp Ile

385 390 395 400385 390 395 400

LysLys

<210> 42<210> 42

<211> 405<211> 405

<212> PRT<212> PRT

<213> 猫咽支原体（Mycoplasma felifaucium）<213> Mycoplasma felifaucium

<400> 42<400> 42

Met Asn Lys Ile Asn Val Tyr Ser Glu Ile Gly Lys Leu Lys Glu ValMet Asn Lys Ile Asn Val Tyr Ser Glu Ile Gly Lys Leu Lys Glu Val

1 5 10 151 5 10 15

Leu Val His Thr Pro Gly Asn Glu Ile Arg Arg Ile Ser Pro Ser ArgLeu Val His Thr Pro Gly Asn Glu Ile Arg Arg Ile Ser Pro Ser Arg

20 25 30 20 25 30

Leu Asp Glu Leu Leu Phe Ser Ala Leu Leu Glu Pro Asn Phe Ala AlaLeu Asp Glu Leu Leu Phe Ser Ala Leu Leu Glu Pro Asn Phe Ala Ala

35 40 45 35 40 45

Lys Glu His Thr Ala Phe Cys Glu Ile Leu Lys Glu Asn Gly Ile LysLys Glu His Thr Ala Phe Cys Glu Ile Leu Lys Glu Asn Gly Ile Lys

50 55 60 50 55 60

Ala Ile Gln Leu Val Asp Leu Val Ser Asp Thr Trp Arg Ile Ala SerAla Ile Gln Leu Val Asp Leu Val Ser Asp Thr Trp Arg Ile Ala Ser

65 70 75 8065 70 75 80

Glu Lys Ala Lys Thr Glu Phe Ile Glu Arg Trp Leu Asp Glu Cys GluGlu Lys Ala Lys Thr Glu Phe Ile Glu Arg Trp Leu Asp Glu Cys Glu

85 90 95 85 90 95

Pro Lys Leu Asp Ser Asn Leu Arg Glu Ile Val Arg Lys His Ile TyrPro Lys Leu Asp Ser Asn Leu Arg Glu Ile Val Arg Lys His Ile Tyr

100 105 110 100 105 110

Ala Ile Glu Lys Arg Ser Val Lys Arg Met Val Lys Thr Met Met AlaAla Ile Glu Lys Arg Ser Val Lys Arg Met Val Lys Thr Met Met Ala

115 120 125 115 120 125

Gly Ile Glu Arg Arg Glu Leu Pro Val Thr Ser Lys Glu Val Ala ArgGly Ile Glu Arg Arg Glu Leu Pro Val Thr Ser Lys Glu Val Ala Arg

130 135 140 130 135 140

Glu Leu Val Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp ProGlu Leu Val Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro

145 150 155 160145 150 155 160

Phe Ala Ser Val Gly Asn Gly Ile Ser Leu His His Met Lys Tyr ValPhe Ala Ser Val Gly Asn Gly Ile Ser Leu His His Met Lys Tyr Val

165 170 175 165 170 175

Thr Arg Gln Arg Glu Thr Ile Phe Ala Glu Phe Val Phe Gly Asn HisThr Arg Gln Arg Glu Thr Ile Phe Ala Glu Phe Val Phe Gly Asn His

180 185 190 180 185 190

Pro Asp Tyr Ile Asp Thr Pro Arg Trp Phe Asp Arg Ser Asp Asp GlyPro Asp Tyr Ile Asp Thr Pro Arg Trp Phe Asp Arg Ser Asp Asp Gly

195 200 205 195 200 205

Arg Ile Glu Gly Gly Asp Val Phe Ile Tyr Gly Ser Lys Thr Leu ValArg Ile Glu Gly Gly Asp Val Phe Ile Tyr Gly Ser Lys Thr Leu Val

210 215 220 210 215 220

Ile Gly Val Ser Glu Arg Thr Asn Lys Glu Ala Ile Lys Val Met AlaIle Gly Val Ser Glu Arg Thr Asn Lys Glu Ala Ile Lys Val Met Ala

225 230 235 240225 230 235 240

Lys Lys Ile Gln Ala Asn Lys Glu Ala Thr Phe Glu Lys Ile Tyr AlaLys Lys Ile Gln Ala Asn Lys Glu Ala Thr Phe Glu Lys Ile Tyr Ala

245 250 255 245 250 255

Ile Asn Val Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp LeuIle Asn Val Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu

260 265 270 260 265 270

Thr Met Leu Asp Lys Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu AlaThr Met Leu Asp Lys Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ala

275 280 285 275 280 285

Val Leu Gln Val Trp Glu Ile Asp Leu Lys Asp Pro Glu Leu Thr TrpVal Leu Gln Val Trp Glu Ile Asp Leu Lys Asp Pro Glu Leu Thr Trp

290 295 300 290 295 300

His Glu Leu Ser Gly Ser Leu Glu Glu Ile Leu His Lys Ile Ile GlyHis Glu Leu Ser Gly Ser Leu Glu Glu Ile Leu His Lys Ile Ile Gly

305 310 315 320305 310 315 320

Arg Lys Pro Ile Leu Ile Pro Ile Ala Gly His Gly Ala Gln Gln IleArg Lys Pro Ile Leu Ile Pro Ile Ala Gly His Gly Ala Gln Gln Ile

325 330 335 325 330 335

Asp Ile Asp Ile Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Ala IleAsp Ile Asp Ile Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Ala Ile

340 345 350 340 345 350

Ala Pro Gly Val Val Val Gly Tyr Asn Arg Asn Val Leu Thr Glu ArgAla Pro Gly Val Val Val Gly Tyr Asn Arg Asn Val Leu Thr Glu Arg

355 360 365 355 360 365

Ala Leu Lys Lys Ala Gly Ile Lys Val Leu Ser Phe Glu Gly Asn GlnAla Leu Lys Lys Ala Gly Ile Lys Val Leu Ser Phe Glu Gly Asn Gln

370 375 380 370 375 380

Leu Ser Leu Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu IleLeu Ser Leu Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Ile

385 390 395 400385 390 395 400

Arg Glu Asn Leu LysArg Glu Asn Leu Lys

405 405

<210> 43<210> 43

<211> 404<211> 404

<212> PRT<212> PRT

<213> 模仿支原体（Mycoplasma imitans）<213> Mycoplasma imitans

<400> 43<400> 43

Met Phe Asn Lys Ile Lys Val Tyr Ser Glu Ile Gly Arg Leu Arg LysMet Phe Asn Lys Ile Lys Val Tyr Ser Glu Ile Gly Arg Leu Arg Lys

1 5 10 151 5 10 15

Val Leu Val His Thr Pro Gly Lys Glu Leu Glu Tyr Val Thr Pro GlnVal Leu Val His Thr Pro Gly Lys Glu Leu Glu Tyr Val Thr Pro Gln

20 25 30 20 25 30

Arg Leu Asp Glu Leu Leu Phe Ser Ser Leu Leu Asn Pro Val Lys AlaArg Leu Asp Glu Leu Leu Phe Ser Ser Leu Leu Asn Pro Val Lys Ala

35 40 45 35 40 45

Arg Gln Glu His Glu Ala Phe Ile Lys Ile Leu Gln Asp Gln Gly ValArg Gln Glu His Glu Ala Phe Ile Lys Ile Leu Gln Asp Gln Gly Val

50 55 60 50 55 60

Glu Cys Val Gln Leu Thr Thr Leu Thr Ala Gln Thr Phe Gln Ser AlaGlu Cys Val Gln Leu Thr Thr Leu Thr Ala Gln Thr Phe Gln Ser Ala

65 70 75 8065 70 75 80

Thr Ser Glu Val Lys Glu Lys Phe Ile Asn Arg Trp Leu Asp Glu CysThr Ser Glu Val Lys Glu Lys Phe Ile Asn Arg Trp Leu Asp Glu Cys

85 90 95 85 90 95

Leu Pro Lys Leu Ser Asp Asp Asn Arg Ile Lys Val Tyr Ala Tyr LeuLeu Pro Lys Leu Ser Asp Asp Asn Arg Ile Lys Val Tyr Ala Tyr Leu

100 105 110 100 105 110

Lys Asp Leu Ser Ser Asp Pro Glu Val Met Ile Arg Lys Met Met SerLys Asp Leu Ser Ser Asp Pro Glu Val Met Ile Arg Lys Met Met Ser

115 120 125 115 120 125

Gly Ile Leu Ala Lys Glu Val Asn Val Gln Ser Asp Val Glu Leu IleGly Ile Leu Ala Lys Glu Val Asn Val Gln Ser Asp Val Glu Leu Ile

130 135 140 130 135 140

Ala Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala SerAla Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser

145 150 155 160145 150 155 160

Ile Gly Lys Gly Val Thr Leu His Ser Met Phe His Pro Thr Arg LysIle Gly Lys Gly Val Thr Leu His Ser Met Phe His Pro Thr Arg Lys

165 170 175 165 170 175

Arg Glu Thr Ile Phe Ala Asp Phe Val Phe Ser His His Pro Glu TyrArg Glu Thr Ile Phe Ala Asp Phe Val Phe Ser His His Pro Glu Tyr

180 185 190 180 185 190

Lys Gln Thr Pro Lys Tyr Tyr Ser Arg Leu Asn Glu Tyr Ser Ile GluLys Gln Thr Pro Lys Tyr Tyr Ser Arg Leu Asn Glu Tyr Ser Ile Glu

195 200 205 195 200 205

Gly Gly Asp Leu Phe Val Tyr Asp Asp Lys Thr Leu Val Ile Gly ValGly Gly Asp Leu Phe Val Tyr Asp Asp Lys Thr Leu Val Ile Gly Val

210 215 220 210 215 220

Ser Glu Arg Thr Glu Lys Lys Ala Ile Gln Phe Leu Ala Glu Lys LeuSer Glu Arg Thr Glu Lys Lys Ala Ile Gln Phe Leu Ala Glu Lys Leu

225 230 235 240225 230 235 240

Arg Glu Asn Tyr Glu Thr Thr Phe Glu Lys Ile Tyr Ala Ile Asn ValArg Glu Asn Tyr Glu Thr Thr Phe Glu Lys Ile Tyr Ala Ile Asn Val

245 250 255 245 250 255

Pro Lys Met Ser Asn Leu Met His Leu Asp Thr Trp Leu Thr Met LeuPro Lys Met Ser Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Leu

260 265 270 260 265 270

Asp Tyr Asp Lys Phe Leu Tyr Ser Pro Asn Met Met Gly Val Leu LysAsp Tyr Asp Lys Phe Leu Tyr Ser Pro Asn Met Met Gly Val Leu Lys

275 280 285 275 280 285

Ile Trp Glu Ile Asp Leu Thr His Glu Gln Leu Ser Trp Arg Glu LeuIle Trp Glu Ile Asp Leu Thr His Glu Gln Leu Ser Trp Arg Glu Leu

290 295 300 290 295 300

Asn Glu Ser Leu Glu Glu Phe Leu Ser Met Val Ile Gly Lys Lys AlaAsn Glu Ser Leu Glu Glu Phe Leu Ser Met Val Ile Gly Lys Lys Ala

305 310 315 320305 310 315 320

Thr Thr Ile Pro Val Ala Gly Glu Asp Ser Thr Gln Ile Glu Ile AspThr Thr Ile Pro Val Ala Gly Glu Asp Ser Thr Gln Ile Glu Ile Asp

325 330 335 325 330 335

Val Glu Thr Asn Phe Asp Ala Thr Asn Phe Leu Val Ile Gln Pro GlyVal Glu Thr Asn Phe Asp Ala Thr Asn Phe Leu Val Ile Gln Pro Gly

340 345 350 340 345 350

Val Val Val Gly Tyr Asp Arg Asn Tyr Lys Thr Asn Gln Ala Leu ValVal Val Val Gly Tyr Asp Arg Asn Tyr Lys Thr Asn Gln Ala Leu Val

355 360 365 355 360 365

Asn Ala Gly Ile Lys Val Leu Ser Trp Asn Gly Asp Gln Leu Ser LeuAsn Ala Gly Ile Lys Val Leu Ser Trp Asn Gly Asp Gln Leu Ser Leu

370 375 380 370 375 380

Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Tyr Arg Asp ProGly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Tyr Arg Asp Pro

385 390 395 400385 390 395 400

Ile Lys Lys GlyIle Lys Lys Gly

<210> 44<210> 44

<211> 401<211> 401

<212> PRT<212> PRT

<213> 乳白色支原体（Mycoplasma opalescens）<213> Mycoplasma opalescens

<400> 44<400> 44

Met Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Thr Leu Lys Glu ValMet Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Thr Leu Lys Glu Val

1 5 10 151 5 10 15

Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Val Ala Pro Ala ArgLeu Val His Thr Pro Gly Asp Glu Ile Arg Arg Val Ala Pro Ala Arg

20 25 30 20 25 30

Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Asn His Ala IleLeu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Asn His Ala Ile

35 40 45 35 40 45

Ala Glu His Lys Ala Phe Ile Lys Ile Leu Glu Asp Asn Gly Ile LysAla Glu His Lys Ala Phe Ile Lys Ile Leu Glu Asp Asn Gly Ile Lys

50 55 60 50 55 60

Val Ile Gln Leu Asp Glu Leu Val Val Gln Thr Trp Asn Gln Val AspVal Ile Gln Leu Asp Glu Leu Val Val Gln Thr Trp Asn Gln Val Asp

65 70 75 8065 70 75 80

Glu Ala Thr Arg Lys Ala Phe Val Thr Lys Trp Leu Asp Glu Cys GluGlu Ala Thr Arg Lys Ala Phe Val Thr Lys Trp Leu Asp Glu Cys Glu

85 90 95 85 90 95

Pro Lys Leu Glu Ser Asn Val Arg Val Glu Val Glu Lys Tyr Ile TyrPro Lys Leu Glu Ser Asn Val Arg Val Glu Val Glu Lys Tyr Ile Tyr

100 105 110 100 105 110

Ser Leu Ala Lys Glu Pro Lys Lys Met Val Arg Thr Met Met Ala GlySer Leu Ala Lys Glu Pro Lys Lys Met Val Arg Thr Met Met Ala Gly

115 120 125 115 120 125

Ile Ser Lys Glu Glu Leu Pro Leu Asn Val Asn Arg Pro Leu Val ValIle Ser Lys Glu Glu Leu Pro Leu Asn Val Asn Arg Pro Leu Val Val

130 135 140 130 135 140

Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser ValAsp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser Val

145 150 155 160145 150 155 160

Gly Thr Gly Ile Ser Leu His His Met Lys Tyr Val Thr Arg Gln ArgGly Thr Gly Ile Ser Leu His His Met Lys Tyr Val Thr Arg Gln Arg

165 170 175 165 170 175

Glu Thr Ile Phe Ala Gln Phe Val Phe Asp Asn His Lys Asp Tyr AsnGlu Thr Ile Phe Ala Gln Phe Val Phe Asp Asn His Lys Asp Tyr Asn

180 185 190 180 185 190

Thr Val Pro Arg Trp Phe Asp Asn Lys Asp Gln Gly Arg Ile Glu GlyThr Val Pro Arg Trp Phe Asp Asn Lys Asp Gln Gly Arg Ile Glu Gly

195 200 205 195 200 205

Gly Asp Val Phe Ile Tyr Asn Thr Lys Thr Leu Val Ile Gly Val SerGly Asp Val Phe Ile Tyr Asn Thr Lys Thr Leu Val Ile Gly Val Ser

210 215 220 210 215 220

Glu Arg Thr Asp Lys Asp Ala Ile Lys Ile Met Ala Lys Lys Ile GlnGlu Arg Thr Asp Lys Asp Ala Ile Lys Ile Met Ala Lys Lys Ile Gln

225 230 235 240225 230 235 240

Ala Asp Lys Asn Cys Lys Phe Glu Lys Ile Phe Ala Ile Asn Val ProAla Asp Lys Asn Cys Lys Phe Glu Lys Ile Phe Ala Ile Asn Val Pro

245 250 255 245 250 255

Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Val AspPro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Val Asp

260 265 270 260 265 270

Arg Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu Lys ValArg Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu Lys Val

275 280 285 275 280 285

Trp Glu Ile Asp Leu Lys Asp Ala Ser Leu Ala Trp Lys Glu Ile GluTrp Glu Ile Asp Leu Lys Asp Ala Ser Leu Ala Trp Lys Glu Ile Glu

290 295 300 290 295 300

Gly Ser Leu Ser Gln Ile Leu Glu Lys Ile Ile Gly Glu Lys Pro IleGly Ser Leu Ser Gln Ile Leu Glu Lys Ile Ile Gly Glu Lys Pro Ile

305 310 315 320305 310 315 320

Leu Ile Pro Ile Ala Gly Glu Asn Ala Ser Gln Leu Asp Ile Asp IleLeu Ile Pro Ile Ala Gly Glu Asn Ala Ser Gln Leu Asp Ile Asp Ile

325 330 335 325 330 335

Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly ValGlu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly Val

340 345 350 340 345 350

Val Val Gly Tyr Ser Arg Asn Val Lys Thr Glu Gln Ala Leu Lys AlaVal Val Gly Tyr Ser Arg Asn Val Lys Thr Glu Gln Ala Leu Lys Ala

355 360 365 355 360 365

Ala Gly Val Lys Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu GlyAla Gly Val Lys Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu Gly

370 375 380 370 375 380

Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Ile Arg Glu Asp LeuMet Gly Ser Ala Arg Cys Met Ser Met Pro Leu Ile Arg Glu Asp Leu

385 390 395 400385 390 395 400

LysLys

<210> 45<210> 45

<211> 425<211> 425

<212> PRT<212> PRT

<213> 毛氏支原体（Mycoplasma moatsii）<213> Mycoplasma moatsii

<400> 45<400> 45

Met Lys Lys Asn Ala Ile Asn Val Tyr Ser Glu Ile Gly Lys Leu LysMet Lys Lys Asn Ala Ile Asn Val Tyr Ser Glu Ile Gly Lys Leu Lys

1 5 10 151 5 10 15

Lys Val Leu Val His Arg Pro Gly Asp Glu Leu Lys Tyr Val Thr ProLys Val Leu Val His Arg Pro Gly Asp Glu Leu Lys Tyr Val Thr Pro

20 25 30 20 25 30

Gln Arg Met Asp Glu Leu Leu Met Ser Ala Ile Ile Glu Leu Glu GlnGln Arg Met Asp Glu Leu Leu Met Ser Ala Ile Ile Glu Leu Glu Gln

35 40 45 35 40 45

Ala Lys Glu Glu His Asp Ala Phe Thr Lys Ile Leu Arg Asp Asn GlyAla Lys Glu Glu His Asp Ala Phe Thr Lys Ile Leu Arg Asp Asn Gly

50 55 60 50 55 60

Val Glu Val Ile Glu Leu Ala Asp Leu Thr Ala Glu Met Tyr Asp SerVal Glu Val Ile Glu Leu Ala Asp Leu Thr Ala Glu Met Tyr Asp Ser

65 70 75 8065 70 75 80

Leu Thr Pro Ser Glu Lys Asp Ala Phe Leu Asn Gln Trp Val Lys GluLeu Thr Pro Ser Glu Lys Asp Ala Phe Leu Asn Gln Trp Val Lys Glu

85 90 95 85 90 95

Ala Ser Trp Gly Lys Lys Ser Ser Ile Asp Ala Leu Lys Ile Lys LysAla Ser Trp Gly Lys Lys Ser Ser Ile Asp Ala Leu Lys Ile Lys Lys

100 105 110 100 105 110

Asn Leu Ser Lys Lys Val Phe Asp Tyr Val Lys Ser Ile Lys Pro ThrAsn Leu Ser Lys Lys Lys Val Phe Asp Tyr Val Lys Ser Ile Lys Pro Thr

115 120 125 115 120 125

Arg Lys Met Ile Asp Lys Leu Met Ala Gly Val Leu Leu Ser Glu IleArg Lys Met Ile Asp Lys Leu Met Ala Gly Val Leu Leu Ser Glu Ile

130 135 140 130 135 140

Gly Glu Lys Ser Ile Ile Leu Asn Lys Asp Lys Lys Asn Glu Met ValGly Glu Lys Ser Ile Ile Leu Asn Lys Asp Lys Lys Asn Glu Met Val

145 150 155 160145 150 155 160

Ile Asp Leu Val Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg AspIle Asp Leu Val Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp

165 170 175 165 170 175

Pro Phe Ala Ser Val Gly Asn Gly Ile Thr Leu His Asn Met Lys TyrPro Phe Ala Ser Val Gly Asn Gly Ile Thr Leu His Asn Met Lys Tyr

180 185 190 180 185 190

Pro Thr Arg Lys Arg Glu Thr Ile Phe Ala Gln Trp Ile Phe Asn LysPro Thr Arg Lys Arg Glu Thr Ile Phe Ala Gln Trp Ile Phe Asn Lys

195 200 205 195 200 205

His Pro Glu Tyr Lys Asp Val Pro Gln Phe Ile Ser Lys Arg Asp GlyHis Pro Glu Tyr Lys Asp Val Pro Gln Phe Ile Ser Lys Arg Asp Gly

210 215 220 210 215 220

Lys Glu Thr Ile Glu Gly Gly Asp Val Phe Ile Tyr Thr Lys Asp ValLys Glu Thr Ile Glu Gly Gly Asp Val Phe Ile Tyr Thr Lys Asp Val

225 230 235 240225 230 235 240

Leu Ala Ile Gly Val Ser Glu Arg Thr Asn Met Glu Ala Ile Leu ArgLeu Ala Ile Gly Val Ser Glu Arg Thr Asn Met Glu Ala Ile Leu Arg

245 250 255 245 250 255

Ile Ala Thr Asn Ile Lys Lys Asp Lys Asn Cys Glu Phe Lys Lys IleIle Ala Thr Asn Ile Lys Lys Asp Lys Asn Cys Glu Phe Lys Lys Ile

260 265 270 260 265 270

Val Ala Ile Asn Val Pro Pro Met Gly Asn Leu Met His Leu Asp ThrVal Ala Ile Asn Val Pro Pro Met Gly Asn Leu Met His Leu Asp Thr

275 280 285 275 280 285

Trp Leu Thr Met Leu Asp Lys Asp Leu Phe Leu Tyr Ser Gly Asn IleTrp Leu Thr Met Leu Asp Lys Asp Leu Phe Leu Tyr Ser Gly Asn Ile

290 295 300 290 295 300

Lys Ser Ala Leu Lys Val Trp Glu Ile Asp Leu Thr Lys Pro Ile ThrLys Ser Ala Leu Lys Val Trp Glu Ile Asp Leu Thr Lys Pro Ile Thr

305 310 315 320305 310 315 320

Pro Lys Ser Pro Lys Leu Ser Thr Ala Lys Leu Ala Asp Ile Leu AlaPro Lys Ser Pro Lys Leu Ser Thr Ala Lys Leu Ala Asp Ile Leu Ala

325 330 335 325 330 335

Lys Ile Val Gly Lys Lys Val Arg Met Ile Pro Ile Gly Gly Lys AspLys Ile Val Gly Lys Lys Val Arg Met Ile Pro Ile Gly Gly Lys Asp

340 345 350 340 345 350

Gly Asn Gln Met Asp Ile Asp Ile Glu Thr His Phe Asp Gly Thr AsnGly Asn Gln Met Asp Ile Asp Ile Glu Thr His Phe Asp Gly Thr Asn

355 360 365 355 360 365

Tyr Leu Ala Ile Ala Pro Gly Val Val Val Gly Tyr His Arg Asn ArgTyr Leu Ala Ile Ala Pro Gly Val Val Val Gly Tyr His Arg Asn Arg

370 375 380 370 375 380

Lys Thr Gln Lys Ala Leu Glu Glu Ala Gly Val Lys Val Leu Ala PheLys Thr Gln Lys Ala Leu Glu Glu Ala Gly Val Lys Val Leu Ala Phe

385 390 395 400385 390 395 400

Gln Gly Asn Gln Leu Ser Leu Gly Met Gly Ser Ala Arg Cys Met SerGln Gly Asn Gln Leu Ser Leu Gly Met Gly Ser Ala Arg Cys Met Ser

405 410 415 405 410 415

Met Pro Leu Val Arg Glu Glu Val LysMet Pro Leu Val Arg Glu Glu Val Lys

420 425 420 425

<210> 46<210> 46

<211> 399<211> 399

<212> PRT<212> PRT

<213> 象支原体（Mycoplasma elephantis）<213> Mycoplasma elephantis

<400> 46<400> 46

Met Ser Gln Ile Asn Val Phe Ser Glu Ile Gly Gln Leu Lys Glu ValMet Ser Gln Ile Asn Val Phe Ser Glu Ile Gly Gln Leu Lys Glu Val

1 5 10 151 5 10 15

Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Lys ArgLeu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Lys Arg

20 25 30 20 25 30

Tyr Asn Glu Leu Leu Phe Ser Ala Ile Leu Glu Ala Asp Val Ala IleTyr Asn Glu Leu Leu Phe Ser Ala Ile Leu Glu Ala Asp Val Ala Ile

35 40 45 35 40 45

Lys Glu His Lys Ser Phe Val Lys Ile Leu Glu Glu Asn Asn Val LysLys Glu His Lys Ser Phe Val Lys Ile Leu Glu Glu Asn Asn Val Lys

50 55 60 50 55 60

Val Ile Gln Leu Lys Asp Ile Leu Leu Glu Thr Trp Asn Ile Cys SerVal Ile Gln Leu Lys Asp Ile Leu Leu Glu Thr Trp Asn Ile Cys Ser

65 70 75 8065 70 75 80

Lys Glu Ala Lys Asn Ile Phe Ile Asn Lys Trp Ile Glu Glu Ala GlnLys Glu Ala Lys Asn Ile Phe Ile Asn Lys Trp Ile Glu Glu Ala Gln

85 90 95 85 90 95

Pro Val Ile His Ser Ser Ser Leu Lys Glu Lys Ile Lys Leu Phe LeuPro Val Ile His Ser Ser Ser Leu Lys Glu Lys Ile Lys Leu Phe Leu

100 105 110 100 105 110

Lys Ser Lys Thr Pro Leu Glu Ile Ile Asp Ile Met Met Lys Gly IleLys Ser Lys Thr Pro Leu Glu Ile Ile Asp Ile Met Met Lys Gly Ile

115 120 125 115 120 125

Leu Lys Gln Glu Leu Gly Ile Glu Tyr Lys His Glu Leu Ile Ile AspLeu Lys Gln Glu Leu Gly Ile Glu Tyr Lys His Glu Leu Ile Ile Asp

130 135 140 130 135 140

Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Thr Ser Met GlyPro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Thr Ser Met Gly

145 150 155 160145 150 155 160

Ser Gly Ile Thr Ile Asn Asn Met Lys Tyr Gln Thr Arg Lys Arg GluSer Gly Ile Thr Ile Asn Asn Met Lys Tyr Gln Thr Arg Lys Arg Glu

165 170 175 165 170 175

Thr Ile Phe Ser Glu Phe Ile Phe Asn Asn His Pro Lys Tyr Lys AsnThr Ile Phe Ser Glu Phe Ile Phe Asn Asn His Pro Lys Tyr Lys Asn

180 185 190 180 185 190

Thr Pro Arg Trp Phe Asp Arg Phe Asp Ser Gly Asn Ile Glu Gly GlyThr Pro Arg Trp Phe Asp Arg Phe Asp Ser Gly Asn Ile Glu Gly Gly

195 200 205 195 200 205

Asp Leu Phe Val Tyr Thr Lys Glu Thr Ile Val Val Gly Val Ser GluAsp Leu Phe Val Tyr Thr Lys Glu Thr Ile Val Val Gly Val Ser Glu

210 215 220 210 215 220

Arg Thr Lys Lys Lys Ala Ile Leu Lys Ile Ala Lys Asn Ile Gln GluArg Thr Lys Lys Lys Lys Ala Ile Leu Lys Ile Ala Lys Asn Ile Gln Glu

225 230 235 240225 230 235 240

Asn Asn Asn Ser Phe Lys Lys Ile Val Val Ile Lys Val Pro Ile MetAsn Asn Asn Ser Phe Lys Lys Ile Val Val Ile Lys Val Pro Ile Met

245 250 255 245 250 255

Gln Asn Leu Met His Leu Asp Thr Trp Ile Val Met Val Asp Phe AspGln Asn Leu Met His Leu Asp Thr Trp Ile Val Met Val Asp Phe Asp

260 265 270 260 265 270

Lys Phe Ile Tyr Ser Pro Asn Val Thr Lys Ser Leu Lys Phe Trp GluLys Phe Ile Tyr Ser Pro Asn Val Thr Lys Ser Leu Lys Phe Trp Glu

275 280 285 275 280 285

Ile Asp Leu Thr Lys Lys Pro Lys Phe Ile Gln Leu Lys Asn Glu ThrIle Asp Leu Thr Lys Lys Pro Lys Phe Ile Gln Leu Lys Asn Glu Thr

290 295 300 290 295 300

Leu Glu Asp Val Leu Tyr Arg Val Ile Gly Lys Lys Pro Ile Leu IleLeu Glu Asp Val Leu Tyr Arg Val Ile Gly Lys Lys Pro Ile Leu Ile

305 310 315 320305 310 315 320

Pro Val Ala Gly Glu Asn Ala Asn Gln Ile Asp Ile Asp Val Glu ThrPro Val Ala Gly Glu Asn Ala Asn Gln Ile Asp Ile Asp Val Glu Thr

325 330 335 325 330 335

His Phe Asp Ala Thr Asn Tyr Leu Thr Ile Arg Pro Gly Val Val ValHis Phe Asp Ala Thr Asn Tyr Leu Thr Ile Arg Pro Gly Val Val Val

340 345 350 340 345 350

Gly Tyr Ser Arg Asn Lys Lys Thr Glu Glu Ala Leu Ile Asn Ala GlyGly Tyr Ser Arg Asn Lys Lys Thr Glu Glu Ala Leu Ile Asn Ala Gly

355 360 365 355 360 365

Val Lys Val Tyr Ala Phe Glu Gly Asn Gln Leu Ser Leu Gly Met GlyVal Lys Val Tyr Ala Phe Glu Gly Asn Gln Leu Ser Leu Gly Met Gly

370 375 380 370 375 380

Ser Ala Arg Cys Met Ser Met Pro Leu Ile Arg Glu Asp Ile IleSer Ala Arg Cys Met Ser Met Pro Leu Ile Arg Glu Asp Ile Ile

385 390 395385 390 395

<210> 47<210> 47

<211> 399<211> 399

<212> PRT<212> PRT

<213> 龟支原体（Mycoplasma testudinis）<213> Mycoplasma testudinis

<400> 47<400> 47

Met Lys Asn Ile Asn Val Tyr Ser Glu Val Gly Lys Leu Lys Glu ValMet Lys Asn Ile Asn Val Tyr Ser Glu Val Gly Lys Leu Lys Glu Val

1 5 10 151 5 10 15

Val Val His Thr Pro Gly Glu Glu Leu His Asn Val Ala Pro Ser ArgVal Val His Thr Pro Gly Glu Glu Leu His Asn Val Ala Pro Ser Arg

20 25 30 20 25 30

Leu Gln Glu Leu Leu Thr Ser Ala Val Leu Glu Pro Glu Val Ala ArgLeu Gln Glu Leu Leu Thr Ser Ala Val Leu Glu Pro Glu Val Ala Arg

35 40 45 35 40 45

Lys Glu His Leu Lys Phe Ile Lys Ile Leu Asn Asp Tyr Gly Val LysLys Glu His Leu Lys Phe Ile Lys Ile Leu Asn Asp Tyr Gly Val Lys

50 55 60 50 55 60

Val Ile Gln Ile Val Asp Leu Ile Thr Glu Thr Tyr Glu Ala Val AspVal Ile Gln Ile Val Asp Leu Ile Thr Glu Thr Tyr Glu Ala Val Asp

65 70 75 8065 70 75 80

Ser Asn Lys Lys Glu Ala Phe Ile Asn Asn Trp Leu Asp Asn Ser ValSer Asn Lys Lys Glu Ala Phe Ile Asn Asn Trp Leu Asp Asn Ser Val

85 90 95 85 90 95

Pro Lys Leu Thr Asp Lys Asn Arg Met Ile Leu Arg Asn Tyr Leu ThrPro Lys Leu Thr Asp Lys Asn Arg Met Ile Leu Arg Asn Tyr Leu Thr

100 105 110 100 105 110

Gln Phe Ser Thr Lys Ala Met Ile Arg Lys Met Ile Ser Gly Ile ArgGln Phe Ser Thr Lys Ala Met Ile Arg Lys Met Ile Ser Gly Ile Arg

115 120 125 115 120 125

Ala Lys Glu Leu Asn Leu Lys Thr Pro Ser Ala Leu Leu Val Asp ProAla Lys Glu Leu Asn Leu Lys Thr Pro Ser Ala Leu Leu Val Asp Pro

130 135 140 130 135 140

Met Pro Asn Leu Cys Phe Ala Arg Asp Thr Phe Ala Cys Val Gly SerMet Pro Asn Leu Cys Phe Ala Arg Asp Thr Phe Ala Cys Val Gly Ser

145 150 155 160145 150 155 160

Ala Ile Ser Leu Ser Thr Met Lys His Pro Thr Arg Arg Arg Glu AlaAla Ile Ser Leu Ser Thr Met Lys His Pro Thr Arg Arg Arg Glu Ala

165 170 175 165 170 175

Leu Leu Thr Glu Phe Ile Phe Gln Asn His Pro Lys Tyr Lys Asp ValLeu Leu Thr Glu Phe Ile Phe Gln Asn His Pro Lys Tyr Lys Asp Val

180 185 190 180 185 190

Ile Lys Tyr Phe Asp Ser Lys Asn Ser Lys Ala Thr Ile Glu Gly GlyIle Lys Tyr Phe Asp Ser Lys Asn Ser Lys Ala Thr Ile Glu Gly Gly

195 200 205 195 200 205

Asp Ile Phe Val Tyr Asn Pro Lys Thr Leu Val Val Gly Asn Ser GluAsp Ile Phe Val Tyr Asn Pro Lys Thr Leu Val Val Gly Asn Ser Glu

210 215 220 210 215 220

Arg Thr Asn Met Gln Ala Cys Leu Leu Leu Ala Lys Lys Ile Gln SerArg Thr Asn Met Gln Ala Cys Leu Leu Leu Ala Lys Lys Ile Gln Ser

225 230 235 240225 230 235 240

Asn Pro Asn Asn Lys Phe Glu Lys Ile Val Ile Val Asn Val Pro ProAsn Pro Asn Asn Lys Phe Glu Lys Ile Val Ile Val Asn Val Pro Pro

245 250 255 245 250 255

Leu Pro His Leu Met His Leu Asp Thr Trp Leu Thr Met Val Asp TyrLeu Pro His Leu Met His Leu Asp Thr Trp Leu Thr Met Val Asp Tyr

260 265 270 260 265 270

Asp Lys Phe Ile Tyr Ser Pro Asn Ile Leu His Thr Leu Lys Phe TrpAsp Lys Phe Ile Tyr Ser Pro Asn Ile Leu His Thr Leu Lys Phe Trp

275 280 285 275 280 285

Val Ile Asp Leu Lys Lys Arg Lys Leu Glu Ala Val Glu Lys His AsnVal Ile Asp Leu Lys Lys Arg Lys Leu Glu Ala Val Glu Lys His Asn

290 295 300 290 295 300

Thr Leu Lys Ala Met Leu Arg Met Ile Ile Lys Lys Glu Pro Ile LeuThr Leu Lys Ala Met Leu Arg Met Ile Ile Lys Lys Glu Pro Ile Leu

305 310 315 320305 310 315 320

Ile Pro Val Gly Asp Val Gly Ala Asp Gln Leu Asp Ile Asp Leu GluIle Pro Val Gly Asp Val Gly Ala Asp Gln Leu Asp Ile Asp Leu Glu

325 330 335 325 330 335

Thr His Phe Asp Ala Thr Asn Tyr Leu Ala Leu Ala Pro Gly Val ValThr His Phe Asp Ala Thr Asn Tyr Leu Ala Leu Ala Pro Gly Val Val

340 345 350 340 345 350

Val Gly Tyr Asp Arg Asn Ile Lys Thr Gln Arg Ala Leu Glu Lys AlaVal Gly Tyr Asp Arg Asn Ile Lys Thr Gln Arg Ala Leu Glu Lys Ala

355 360 365 355 360 365

Gly Val Lys Val Leu Ser Phe Ser Gly Asn Gln Leu Ser Leu Ala MetGly Val Lys Val Leu Ser Phe Ser Gly Asn Gln Leu Ser Leu Ala Met

370 375 380 370 375 380

Gly Ser Ala Arg Cys Leu Ser Met Pro Leu Ile Arg Glu Glu AsnGly Ser Ala Arg Cys Leu Ser Met Pro Leu Ile Arg Glu Glu Asn

385 390 395385 390 395

<210> 48<210> 48

<211> 410<211> 410

<212> PRT<212> PRT

<213> 加拿大支原体（Mycoplasma canadense）<213> Mycoplasma canadense

<400> 48<400> 48

Met Ser Val Phe Asp Ser Lys Phe Lys Gly Ile His Val Tyr Ser GluMet Ser Val Phe Asp Ser Lys Phe Lys Gly Ile His Val Tyr Ser Glu

1 5 10 151 5 10 15

Ile Gly Glu Leu Glu Ser Val Leu Val His Glu Pro Gly Arg Glu IleIle Gly Glu Leu Glu Ser Val Leu Val His Glu Pro Gly Arg Glu Ile

20 25 30 20 25 30

Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala IleAsp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile

35 40 45 35 40 45

Leu Glu Ser His Asp Ala Arg Lys Glu His Lys Gln Phe Val Ser GluLeu Glu Ser His Asp Ala Arg Lys Glu His Lys Gln Phe Val Ser Glu

50 55 60 50 55 60

Leu Lys Ala Asn Asp Ile Asn Val Val Glu Leu Thr Asp Leu Val AlaLeu Lys Ala Asn Asp Ile Asn Val Val Glu Leu Thr Asp Leu Val Ala

65 70 75 8065 70 75 80

Glu Thr Tyr Asp Leu Ala Ser Gln Glu Ala Lys Asp Lys Leu Ile GluGlu Thr Tyr Asp Leu Ala Ser Gln Glu Ala Lys Asp Lys Leu Ile Glu

85 90 95 85 90 95

Glu Phe Leu Glu Asp Ser Glu Pro Val Leu Ser Glu Glu His Lys AlaGlu Phe Leu Glu Asp Ser Glu Pro Val Leu Ser Glu Glu His Lys Ala

100 105 110 100 105 110

Ile Val Arg Lys Tyr Leu Lys Gly Ile Gln Pro Thr Arg Lys Leu IleIle Val Arg Lys Tyr Leu Lys Gly Ile Gln Pro Thr Arg Lys Leu Ile

115 120 125 115 120 125

Glu Met Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Glu AlaGlu Met Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Glu Ala

130 135 140 130 135 140

Asp His Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr ArgAsp His Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg

145 150 155 160145 150 155 160

Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met ArgAsp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg

165 170 175 165 170 175

Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Val Phe SerTyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Val Phe Ser

180 185 190 180 185 190

Asn His Pro Lys Leu Val Asn Thr Pro Trp Tyr Tyr Asp Pro Ser LeuAsn His Pro Lys Leu Val Asn Thr Pro Trp Tyr Tyr Asp Pro Ser Leu

195 200 205 195 200 205

Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Val Tyr Asn Asn Asp ThrLys Leu Ser Ile Glu Gly Gly Asp Val Phe Val Tyr Asn Asn Asp Thr

210 215 220 210 215 220

Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Gln Thr Val Thr LeuLeu Val Val Gly Val Ser Glu Arg Thr Asp Leu Gln Thr Val Thr Leu

225 230 235 240225 230 235 240

Leu Ala Lys Asn Ile Val Ala Asn Lys Glu Cys Glu Phe Lys Arg IleLeu Ala Lys Asn Ile Val Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile

245 250 255 245 250 255

Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp ThrVal Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr

260 265 270 260 265 270

Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile AlaTrp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala

275 280 285 275 280 285

Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly SerAsn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ser

290 295 300 290 295 300

Glu Pro Gln Pro Val Glu Asn Gly Leu Pro Leu Glu Gly Leu Leu GluGlu Pro Gln Pro Val Glu Asn Gly Leu Pro Leu Glu Gly Leu Leu Glu

305 310 315 320305 310 315 320

Ser Ile Ile Asn Lys Lys Pro Ile Leu Ile Pro Ile Ala Gly Glu GlySer Ile Ile Asn Lys Lys Pro Ile Leu Ile Pro Ile Ala Gly Glu Gly

325 330 335 325 330 335

Ala Ser Gln Met Glu Ile Glu Arg Glu Thr His Phe Asp Gly Thr AsnAla Ser Gln Met Glu Ile Glu Arg Glu Thr His Phe Asp Gly Thr Asn

340 345 350 340 345 350

Tyr Leu Ala Ile Arg Pro Gly Val Val Ile Gly Tyr Ser Arg Asn GluTyr Leu Ala Ile Arg Pro Gly Val Val Ile Gly Tyr Ser Arg Asn Glu

355 360 365 355 360 365

Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Lys Val Leu Pro PheLys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Lys Val Leu Pro Phe

370 375 380 370 375 380

His Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met SerHis Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser

385 390 395 400385 390 395 400

Met Pro Leu Ser Arg Lys Asp Val Lys TrpMet Pro Leu Ser Arg Lys Asp Val Lys Trp

405 410 405 410

<210> 49<210> 49

<211> 409<211> 409

<212> PRT<212> PRT

<213> 鹅支原体（Mycoplasma anseris）<213> Mycoplasma anseris

<400> 49<400> 49

Met Ser Val Phe Asp Lys Arg Phe Lys Gly Ile His Val Tyr Ser GluMet Ser Val Phe Asp Lys Arg Phe Lys Gly Ile His Val Tyr Ser Glu

1 5 10 151 5 10 15

Ile Gly Glu Leu Gln Thr Val Leu Val His Glu Pro Gly Arg Glu IleIle Gly Glu Leu Gln Thr Val Leu Val His Glu Pro Gly Arg Glu Ile

20 25 30 20 25 30

Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala IleAsp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile

35 40 45 35 40 45

Leu Glu Ser His Asp Ala Arg Ala Glu His Lys Lys Phe Val Ala ThrLeu Glu Ser His Asp Ala Arg Ala Glu His Lys Lys Phe Val Ala Thr

50 55 60 50 55 60

Leu Lys Glu Gln Gly Ile Asn Thr Val Glu Leu Thr Asp Leu Val AlaLeu Lys Glu Gln Gly Ile Asn Thr Val Glu Leu Thr Asp Leu Val Ala

65 70 75 8065 70 75 80

Glu Thr Tyr Asp Leu Ala Ser Gln Glu Ala Arg Asp Asn Leu Leu GluGlu Thr Tyr Asp Leu Ala Ser Gln Glu Ala Arg Asp Asn Leu Leu Glu

85 90 95 85 90 95

Glu Phe Leu Asp Asp Ser Ala Pro Val Leu Ser Glu Glu His Lys GluGlu Phe Leu Asp Asp Ser Ala Pro Val Leu Ser Glu Glu His Lys Glu

100 105 110 100 105 110

Ile Val Arg Thr Tyr Leu Lys Gly Ile Lys Gly Thr Arg Lys Leu IleIle Val Arg Thr Tyr Leu Lys Gly Ile Lys Gly Thr Arg Lys Leu Ile

115 120 125 115 120 125

Glu Thr Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Glu AlaGlu Thr Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Glu Ala

130 135 140 130 135 140

Glu Gln Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr ArgGlu Gln Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg

145 150 155 160145 150 155 160

Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met ArgAsp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg

165 170 175 165 170 175

Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Ile Phe SerTyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Ile Phe Ser

180 185 190 180 185 190

Asn His Pro Gln Leu Val Asn Thr Pro Trp Tyr Tyr Asn Pro Ala GluAsn His Pro Gln Leu Val Asn Thr Pro Trp Tyr Tyr Asn Pro Ala Glu

195 200 205 195 200 205

Gly Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp ThrGly Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr

210 215 220 210 215 220

Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Gln Thr Ile Thr LeuLeu Val Val Gly Val Ser Glu Arg Thr Asp Leu Gln Thr Ile Thr Leu

225 230 235 240225 230 235 240

Leu Ala Lys Asn Ile Lys Ala Asn Glu Glu Cys Glu Phe Lys Arg IleLeu Ala Lys Asn Ile Lys Ala Asn Glu Glu Cys Glu Phe Lys Arg Ile

245 250 255 245 250 255

Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp ThrVal Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr

260 265 270 260 265 270

Trp Leu Thr Met Leu Asp Thr Asn Lys Phe Leu Tyr Ser Pro Ile AlaTrp Leu Thr Met Leu Asp Thr Asn Lys Phe Leu Tyr Ser Pro Ile Ala

275 280 285 275 280 285

Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly AspAsn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Asp

290 295 300 290 295 300

Glu Pro Gln Pro Val Asp Asn Gly Leu Pro Leu Asn Glu Leu Leu LysGlu Pro Gln Pro Val Asp Asn Gly Leu Pro Leu Asn Glu Leu Leu Lys

305 310 315 320305 310 315 320

Ser Ile Ile Gly Glu Glu Pro Ile Leu Ile Pro Ile Ala Gly Asp GlySer Ile Ile Gly Glu Glu Pro Ile Leu Ile Pro Ile Ala Gly Asp Gly

325 330 335 325 330 335

Ala Thr Gln Ile Glu Ile Glu Arg Glu Thr His Phe Asp Gly Thr AsnAla Thr Gln Ile Glu Ile Glu Arg Glu Thr His Phe Asp Gly Thr Asn

340 345 350 340 345 350

Tyr Leu Ala Ile Ala Pro Gly Val Val Ile Gly Tyr Ser Arg Asn GluTyr Leu Ala Ile Ala Pro Gly Val Val Ile Gly Tyr Ser Arg Asn Glu

355 360 365 355 360 365

Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Lys Val Leu Pro PheLys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Lys Val Leu Pro Phe

370 375 380 370 375 380

Lys Gly His Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met SerLys Gly His Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser

385 390 395 400385 390 395 400

Met Pro Leu Tyr Arg Lys Asp Val LysMet Pro Leu Tyr Arg Lys Asp Val Lys

405 405

<210> 50<210> 50

<211> 401<211> 401

<212> PRT<212> PRT

<213> 火鸡支原体（Mycoplasma meleagridis）<213> Mycoplasma meleagridis

<400> 50<400> 50

Met Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Val Leu Lys Glu ValMet Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Val Leu Lys Glu Val

1 5 10 151 5 10 15

Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser ArgLeu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser Arg

20 25 30 20 25 30

Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Gln Pro Glu Gln Ala IleLeu Asp Glu Leu Leu Phe Ser Ala Ile Leu Gln Pro Glu Gln Ala Ile

35 40 45 35 40 45

Lys Glu His Gln Ser Phe Val Lys Ile Leu Gln Asp Arg Gly Ile LysLys Glu His Gln Ser Phe Val Lys Ile Leu Gln Asp Arg Gly Ile Lys

50 55 60 50 55 60

Val Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Val Lys Tyr AlaVal Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Val Lys Tyr Ala

65 70 75 8065 70 75 80

Thr Ser Lys Glu Lys Glu Ser Phe Ile Glu Lys Trp Leu Asp Glu AlaThr Ser Lys Glu Lys Glu Ser Phe Ile Glu Lys Trp Leu Asp Glu Ala

85 90 95 85 90 95

Thr Pro Ala Leu Asn Ser Glu Asn Arg Ala Arg Val Lys Asn Tyr IleThr Pro Ala Leu Asn Ser Glu Asn Arg Ala Arg Val Lys Asn Tyr Ile

100 105 110 100 105 110

Thr Ala Met Gln Gly Gln Pro Val Lys Met Val Arg Ala Met Met AlaThr Ala Met Gln Gly Gln Pro Val Lys Met Val Arg Ala Met Met Ala

115 120 125 115 120 125

Gly Val Ser Lys Gln Glu Leu Asn Ile Glu Ser Asp Val Glu Leu IleGly Val Ser Lys Gln Glu Leu Asn Ile Glu Ser Asp Val Glu Leu Ile

130 135 140 130 135 140

Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala SerVal Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser

145 150 155 160145 150 155 160

Ala Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg LysAla Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg Lys

165 170 175 165 170 175

Arg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ser Ile His Pro Glu TyrArg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ser Ile His Pro Glu Tyr

180 185 190 180 185 190

Lys Gln Thr Pro His Trp Phe Asp Arg Leu Asp Lys Gly Asn Ile GluLys Gln Thr Pro His Trp Phe Asp Arg Leu Asp Lys Gly Asn Ile Glu

195 200 205 195 200 205

Gly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly ValGly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly Val

210 215 220 210 215 220

Ser Glu Arg Thr Asn Lys Glu Ala Ile Leu Thr Ile Ala Glu His IleSer Glu Arg Thr Asn Lys Glu Ala Ile Leu Thr Ile Ala Glu His Ile

225 230 235 240225 230 235 240

Lys Asn Asn Lys Glu Ala Lys Phe Lys Lys Ile Val Ala Ile Asn ValLys Asn Asn Lys Glu Ala Lys Phe Lys Lys Ile Val Ala Ile Asn Val

245 250 255 245 250 255

Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met ValPro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Val

260 265 270 260 265 270

Asp Lys Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu LysAsp Lys Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu Lys

275 280 285 275 280 285

Ile Trp Glu Ile Asp Leu Ser Lys Glu Ile Lys Met Val Glu Thr SerIle Trp Glu Ile Asp Leu Ser Lys Glu Ile Lys Met Val Glu Thr Ser

290 295 300 290 295 300

Lys Pro Leu Ala Asp Val Leu Glu Ser Ile Ile Gly Glu Lys Pro IleLys Pro Leu Ala Asp Val Leu Glu Ser Ile Ile Gly Glu Lys Pro Ile

305 310 315 320305 310 315 320

Leu Ile Pro Ile Ala Gly Glu Asn Ala Ser Gln Leu Asp Ile Asp IleLeu Ile Pro Ile Ala Gly Glu Asn Ala Ser Gln Leu Asp Ile Asp Ile

325 330 335 325 330 335

Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly ValGlu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly Val

340 345 350 340 345 350

Val Val Gly Tyr Ser Arg Asn Val Lys Thr Glu Ala Ala Leu Lys AlaVal Val Gly Tyr Ser Arg Asn Val Lys Thr Glu Ala Ala Leu Lys Ala

355 360 365 355 360 365

Ala Gly Val Thr Val Tyr Ser Phe Asp Gly Asn Gln Leu Ser Leu GlyAla Gly Val Thr Val Tyr Ser Phe Asp Gly Asn Gln Leu Ser Leu Gly

370 375 380 370 375 380

Met Gly Ser Gly Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp ValMet Gly Ser Gly Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp Val

385 390 395 400385 390 395 400

LysLys

<210> 51<210> 51

<211> 404<211> 404

<212> PRT<212> PRT

<213> 肠支原体（Mycoplasma alvi）<213> Mycoplasma alvi

<400> 51<400> 51

Met Ser Ile Lys Glu Asn Gly Ile His Val Tyr Ser Glu Ile Gly LysMet Ser Ile Lys Glu Asn Gly Ile His Val Tyr Ser Glu Ile Gly Lys

1 5 10 151 5 10 15

Leu Arg Asp Val Leu Val His Arg Pro Gly Arg Glu Leu Asn Phe LeuLeu Arg Asp Val Leu Val His Arg Pro Gly Arg Glu Leu Asn Phe Leu

20 25 30 20 25 30

Asp Pro Ser Arg Leu Asp Glu Leu Leu Phe Ala Ala Thr Leu Glu ProAsp Pro Ser Arg Leu Asp Glu Leu Leu Phe Ala Ala Thr Leu Glu Pro

35 40 45 35 40 45

Glu Thr Ala Arg Leu Glu His Asp Asn Phe Thr Thr Val Leu Lys AsnGlu Thr Ala Arg Leu Glu His Asp Asn Phe Thr Thr Val Leu Lys Asn

50 55 60 50 55 60

Gln Gly Val Asn Val Ile Glu Leu Ala Asp Leu Val Ser Gln Thr TyrGln Gly Val Asn Val Ile Glu Leu Ala Asp Leu Val Ser Gln Thr Tyr

65 70 75 8065 70 75 80

Ser Lys Val Asp Ser Lys Val Lys Lys Glu Phe Ile Asp Gln Tyr LeuSer Lys Val Asp Ser Lys Val Lys Lys Glu Phe Ile Asp Gln Tyr Leu

85 90 95 85 90 95

Asn Glu Ala Thr Pro Lys Leu Thr Ser Glu Leu Ser Lys Lys Val TyrAsn Glu Ala Thr Pro Lys Leu Thr Ser Glu Leu Ser Lys Lys Val Tyr

100 105 110 100 105 110

Asp Phe Leu Thr Lys Gln Lys Ser Asn Arg Glu Met Val Asp Phe MetAsp Phe Leu Thr Lys Gln Lys Ser Asn Arg Glu Met Val Asp Phe Met

115 120 125 115 120 125

Met Gly Gly Ile Leu Ser Ser Asp Leu Asn Ile Lys Gly Gln Pro TyrMet Gly Gly Ile Leu Ser Ser Asp Leu Asn Ile Lys Gly Gln Pro Tyr

130 135 140 130 135 140

Leu Ile Val Glu Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro PheLeu Ile Val Glu Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe

145 150 155 160145 150 155 160

Ala Ser Val Gly Asn Gly Ala Thr Ile His Trp Met Lys His Asn ValAla Ser Val Gly Asn Gly Ala Thr Ile His Trp Met Lys His Asn Val

165 170 175 165 170 175

Arg Arg Arg Glu Val Leu Phe Ala Asn Phe Ile Phe Lys Tyr Asn GluArg Arg Arg Glu Val Leu Phe Ala Asn Phe Ile Phe Lys Tyr Asn Glu

180 185 190 180 185 190

Arg Phe Gln Asn Thr Pro Lys Tyr Ile Thr Pro Thr Lys Gly Leu AspArg Phe Gln Asn Thr Pro Lys Tyr Ile Thr Pro Thr Lys Gly Leu Asp

195 200 205 195 200 205

Ile Glu Gly Gly Asp Val Phe Val Tyr Asn Lys Lys Thr Leu Val ValIle Glu Gly Gly Asp Val Phe Val Tyr Asn Lys Lys Thr Leu Val Val

210 215 220 210 215 220

Gly Val Ser Glu Arg Thr Lys Met Glu Thr Ile Lys Glu Leu Ala LysGly Val Ser Glu Arg Thr Lys Met Glu Thr Ile Lys Glu Leu Ala Lys

225 230 235 240225 230 235 240

Asn Ile Ser Lys Asn Lys Glu Cys Thr Phe Thr Lys Ile Tyr Ala IleAsn Ile Ser Lys Asn Lys Glu Cys Thr Phe Thr Lys Ile Tyr Ala Ile

245 250 255 245 250 255

Asn Val Pro Lys Met Pro Asn Leu Met His Leu Asp Thr Trp Leu ThrAsn Val Pro Lys Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr

260 265 270 260 265 270

Met Leu Asp Tyr Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser ValMet Leu Asp Tyr Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val

275 280 285 275 280 285

Leu Lys Val Trp Glu Ile Asn Ile Ser Asn Asn Lys Val Ser Ala ProLeu Lys Val Trp Glu Ile Asn Ile Ser Asn Asn Lys Val Ser Ala Pro

290 295 300 290 295 300

Lys Glu Leu Asn Val Asn Leu Glu Lys Ala Leu Ser Met Ile Ile GlyLys Glu Leu Asn Val Asn Leu Glu Lys Ala Leu Ser Met Ile Ile Gly

305 310 315 320305 310 315 320

Lys Lys Pro Ile Leu Ile Pro Val Ala Gly Ala Asn Ala Ser Gln IleLys Lys Pro Ile Leu Ile Pro Val Ala Gly Ala Asn Ala Ser Gln Ile

325 330 335 325 330 335

Asp Ile Asn Ile Glu Thr Asn Phe Asp Ala Thr Asn Tyr Leu Val IleAsp Ile Asn Ile Glu Thr Asn Phe Asp Ala Thr Asn Tyr Leu Val Ile

340 345 350 340 345 350

Glu Pro Gly Val Val Val Gly Tyr Ser Arg Asn Lys Lys Thr Glu GluGlu Pro Gly Val Val Val Gly Tyr Ser Arg Asn Lys Lys Thr Glu Glu

355 360 365 355 360 365

Ala Leu Val Lys Ala Gly Ile Lys Val Leu Pro Phe His Gly Asn GlnAla Leu Val Lys Ala Gly Ile Lys Val Leu Pro Phe His Gly Asn Gln

370 375 380 370 375 380

Leu Ser Leu Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu TyrLeu Ser Leu Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Tyr

385 390 395 400385 390 395 400

Arg Glu Asp ValArg Glu Asp Val

<210> 52<210> 52

<211> 410<211> 410

<212> PRT<212> PRT

<213> 穿透支原体（Mycoplasma penetrans）<213> Mycoplasma penetrans

<400> 52<400> 52

Met Ser Ser Ile Asp Lys Asn Ser Leu Gly Asn Gly Ile Asn Val TyrMet Ser Ser Ile Asp Lys Asn Ser Leu Gly Asn Gly Ile Asn Val Tyr

1 5 10 151 5 10 15

Ser Glu Ile Gly Glu Leu Lys Glu Val Leu Val His Thr Pro Gly AspSer Glu Ile Gly Glu Leu Lys Glu Val Leu Val His Thr Pro Gly Asp

20 25 30 20 25 30

Glu Ile Arg Tyr Thr Ala Pro Ser Arg Leu Glu Glu Leu Leu Phe SerGlu Ile Arg Tyr Thr Ala Pro Ser Arg Leu Glu Glu Leu Leu Phe Ser

35 40 45 35 40 45

Ala Val Leu Lys Ala Asp Thr Ala Ile Glu Glu His Lys Gly Phe ValAla Val Leu Lys Ala Asp Thr Ala Ile Glu Glu His Lys Gly Phe Val

50 55 60 50 55 60

Lys Ile Leu Gln Asn Asn Gly Ile Lys Val Ile Gln Leu Cys Asp LeuLys Ile Leu Gln Asn Asn Gly Ile Lys Val Ile Gln Leu Cys Asp Leu

65 70 75 8065 70 75 80

Val Ala Glu Thr Tyr Glu Leu Cys Ser Lys Glu Val Arg Asn Ser PheVal Ala Glu Thr Tyr Glu Leu Cys Ser Lys Glu Val Arg Asn Ser Phe

85 90 95 85 90 95

Ile Glu Gln Tyr Leu Asp Glu Ala Leu Pro Val Leu Lys Lys Glu IleIle Glu Gln Tyr Leu Asp Glu Ala Leu Pro Val Leu Lys Lys Glu Ile

100 105 110 100 105 110

Arg Pro Val Val Lys Asp Tyr Leu Leu Ser Phe Pro Thr Val Gln MetArg Pro Val Val Lys Asp Tyr Leu Leu Ser Phe Pro Thr Val Gln Met

115 120 125 115 120 125

Val Arg Lys Met Met Ser Gly Ile Leu Ala Asn Glu Leu Asn Ile LysVal Arg Lys Met Met Ser Gly Ile Leu Ala Asn Glu Leu Asn Ile Lys

130 135 140 130 135 140

Gln Asp Asn Pro Leu Ile Ile Asp Gly Met Pro Asn Leu Tyr Phe ThrGln Asp Asn Pro Leu Ile Ile Asp Gly Met Pro Asn Leu Tyr Phe Thr

145 150 155 160145 150 155 160

Arg Asp Pro Phe Ala Ser Met Gly Asn Gly Val Ser Ile Asn Cys MetArg Asp Pro Phe Ala Ser Met Gly Asn Gly Val Ser Ile Asn Cys Met

165 170 175 165 170 175

Lys Tyr Pro Thr Arg Lys Arg Glu Val Ile Phe Ser Arg Phe Val PheLys Tyr Pro Thr Arg Lys Arg Glu Val Ile Phe Ser Arg Phe Val Phe

180 185 190 180 185 190

Thr Asn Asn Pro Lys Tyr Lys Asn Thr Pro Arg Tyr Phe Asp Ile ValThr Asn Asn Pro Lys Tyr Lys Asn Thr Pro Arg Tyr Phe Asp Ile Val

195 200 205 195 200 205

Gly Asn Asn Gly Thr Ile Glu Gly Gly Asp Ile Phe Ile Tyr Asn SerGly Asn Asn Gly Thr Ile Glu Gly Gly Asp Ile Phe Ile Tyr Asn Ser

210 215 220 210 215 220

Lys Thr Leu Val Ile Gly Asn Ser Glu Arg Thr Asn Phe Ala Ala IleLys Thr Leu Val Ile Gly Asn Ser Glu Arg Thr Asn Phe Ala Ala Ile

225 230 235 240225 230 235 240

Glu Ser Val Ala Lys Asn Ile Gln Ala Asn Lys Asp Cys Thr Phe GluGlu Ser Val Ala Lys Asn Ile Gln Ala Asn Lys Asp Cys Thr Phe Glu

245 250 255 245 250 255

Arg Ile Val Val Ile Asn Val Pro Pro Met Pro Asn Leu Met His LeuArg Ile Val Val Ile Asn Val Pro Pro Met Pro Asn Leu Met His Leu

260 265 270 260 265 270

Asp Thr Trp Leu Thr Met Leu Asp Tyr Asp Lys Phe Leu Tyr Ser ProAsp Thr Trp Leu Thr Met Leu Asp Tyr Asp Lys Phe Leu Tyr Ser Pro

275 280 285 275 280 285

Asn Met Met Asn Val Leu Lys Ile Trp Glu Ile Asp Leu Asn Val LysAsn Met Met Asn Val Leu Lys Ile Trp Glu Ile Asp Leu Asn Val Lys

290 295 300 290 295 300

Pro Val Lys Phe Val Glu Lys Lys Gly Thr Leu Glu Glu Val Leu TyrPro Val Lys Phe Val Glu Lys Lys Lys Gly Thr Leu Glu Glu Val Leu Tyr

305 310 315 320305 310 315 320

Ser Ile Ile Asp Lys Lys Pro Ile Leu Ile Pro Ile Ala Gly Lys GlySer Ile Ile Asp Lys Lys Pro Ile Leu Ile Pro Ile Ala Gly Lys Gly

325 330 335 325 330 335

Ala Asn Gln Leu Asp Ile Asp Ile Glu Thr His Phe Asp Gly Thr AsnAla Asn Gln Leu Asp Ile Asp Ile Glu Thr His Phe Asp Gly Thr Asn

340 345 350 340 345 350

Tyr Leu Thr Ile Ala Pro Gly Val Val Val Gly Tyr Glu Arg Asn GluTyr Leu Thr Ile Ala Pro Gly Val Val Val Gly Tyr Glu Arg Asn Glu

355 360 365 355 360 365

Lys Thr Gln Lys Ala Leu Val Glu Ala Gly Ile Lys Val Leu Ser PheLys Thr Gln Lys Ala Leu Val Glu Ala Gly Ile Lys Val Leu Ser Phe

370 375 380 370 375 380

Asn Gly Ser Gln Leu Ser Leu Gly Met Gly Ser Ala Arg Cys Met SerAsn Gly Ser Gln Leu Ser Leu Gly Met Gly Ser Ala Arg Cys Met Ser

385 390 395 400385 390 395 400

Met Pro Leu Ile Arg Glu Asn Leu Lys LysMet Pro Leu Ile Arg Glu Asn Leu Lys Lys

405 410 405 410

<210> 53<210> 53

<211> 404<211> 404

<212> PRT<212> PRT

<213> 发酵支原体（Mycoplasma fermentans）<213> Mycoplasma fermentans

<400> 53<400> 53

Met Lys Lys Ile Asn Val Tyr Ser Glu Tyr Gly Lys Leu Lys Glu ValMet Lys Lys Ile Asn Val Tyr Ser Glu Tyr Gly Lys Leu Lys Glu Val

1 5 10 151 5 10 15

Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ala Pro Ser ArgLeu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ala Pro Ser Arg

20 25 30 20 25 30

Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Asp Ser Ala IleLeu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Asp Ser Ala Ile

35 40 45 35 40 45

Ala Glu His Lys Arg Phe Val Gln Leu Leu Lys Asp Asn Gly Ile LysAla Glu His Lys Arg Phe Val Gln Leu Leu Lys Asp Asn Gly Ile Lys

50 55 60 50 55 60

Val Ile Gln Leu Asp Glu Leu Phe Ala Lys Thr Phe Asp Leu Val SerVal Ile Gln Leu Asp Glu Leu Phe Ala Lys Thr Phe Asp Leu Val Ser

65 70 75 8065 70 75 80

Glu Ser Val Lys Gln Ser Phe Ile Glu Arg Trp Leu Asp Glu Cys GluGlu Ser Val Lys Gln Ser Phe Ile Glu Arg Trp Leu Asp Glu Cys Glu

85 90 95 85 90 95

Pro Lys Leu Asp Ala Thr Leu Arg Ala Lys Val Lys Glu Tyr Ile LeuPro Lys Leu Asp Ala Thr Leu Arg Ala Lys Val Lys Glu Tyr Ile Leu

100 105 110 100 105 110

Glu Leu Lys Ala Lys Ser Ser Lys Lys Met Val Arg Val Met Met AlaGlu Leu Lys Ala Lys Ser Ser Lys Lys Met Val Arg Val Met Met Ala

115 120 125 115 120 125

Gly Ile Asp Lys Lys Glu Leu Gly Ile Glu Leu Asp Arg Asp Leu ValGly Ile Asp Lys Lys Glu Leu Gly Ile Glu Leu Asp Arg Asp Leu Val

130 135 140 130 135 140

Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala SerVal Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser

145 150 155 160145 150 155 160

Val Gly Asn Gly Ile Ser Leu His His Met Lys Tyr Val Thr Arg GlnVal Gly Asn Gly Ile Ser Leu His His Met Lys Tyr Val Thr Arg Gln

165 170 175 165 170 175

Arg Glu Thr Ile Phe Ser Glu Phe Ile Phe Asp Asn Asn Leu Asp TyrArg Glu Thr Ile Phe Ser Glu Phe Ile Phe Asp Asn Asn Leu Asp Tyr

180 185 190 180 185 190

Asn Thr Val Pro Arg Trp Phe Asp Arg Lys Asp Glu Gly Arg Ile GluAsn Thr Val Pro Arg Trp Phe Asp Arg Lys Asp Glu Gly Arg Ile Glu

195 200 205 195 200 205

Gly Gly Asp Val Phe Ile Tyr Ser Ala Asp Thr Leu Val Val Gly ValGly Gly Asp Val Phe Ile Tyr Ser Ala Asp Thr Leu Val Val Gly Val

210 215 220 210 215 220

Ser Glu Arg Thr Asn Lys Glu Ala Ile Asn Val Met Ala Arg Lys IleSer Glu Arg Thr Asn Lys Glu Ala Ile Asn Val Met Ala Arg Lys Ile

225 230 235 240225 230 235 240

Ala Ala Asp Lys Glu Val Lys Phe Lys Arg Ile Tyr Ala Ile Asn ValAla Ala Asp Lys Glu Val Lys Phe Lys Arg Ile Tyr Ala Ile Asn Val

245 250 255 245 250 255

Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met LeuPro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Leu

260 265 270 260 265 270

Asp Lys Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu LysAsp Lys Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu Lys

275 280 285 275 280 285

Val Trp Arg Ile Asp Leu Asn Asp Pro Asp Phe Val Trp His Glu IleVal Trp Arg Ile Asp Leu Asn Asp Pro Asp Phe Val Trp His Glu Ile

290 295 300 290 295 300

Glu Gly Ser Leu Glu Glu Ile Leu Glu Gln Ile Ile Gly Met Lys ProGlu Gly Ser Leu Glu Glu Ile Leu Glu Gln Ile Ile Gly Met Lys Pro

305 310 315 320305 310 315 320

Ile Leu Ile Pro Ile Ala Gly Lys Gly Ala Ser Gln Leu Asp Ile AspIle Leu Ile Pro Ile Ala Gly Lys Gly Ala Ser Gln Leu Asp Ile Asp

325 330 335 325 330 335

Ile Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro SerIle Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Ser

340 345 350 340 345 350

Val Val Val Gly Tyr Ser Arg Asn Glu Lys Thr Glu Lys Ala Leu LysVal Val Val Gly Tyr Ser Arg Asn Glu Lys Thr Glu Lys Ala Leu Lys

355 360 365 355 360 365

Ala Ala Lys Val Lys Val Leu Ser Phe Glu Gly Asn Gln Leu Ser LeuAla Ala Lys Val Lys Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu

370 375 380 370 375 380

Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Ile Arg Glu AspGly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Ile Arg Glu Asp

385 390 395 400385 390 395 400

Ile Lys Lys LysIle Lys Lys Lys

<210> 54<210> 54

<211> 404<211> 404

<212> PRT<212> PRT

<213> 肺炎支原体（Mycoplasma pneumoniae）<213> Mycoplasma pneumoniae

<400> 54<400> 54

Met Lys Tyr Asn Ile Asn Val His Ser Glu Ile Gly Gln Leu Gln ThrMet Lys Tyr Asn Ile Asn Val His Ser Glu Ile Gly Gln Leu Gln Thr

1 5 10 151 5 10 15

Val Leu Val His Thr Pro Gly Asn Glu Ile Arg Arg Ile Ser Pro ArgVal Leu Val His Thr Pro Gly Asn Glu Ile Arg Arg Ile Ser Pro Arg

20 25 30 20 25 30

Arg Leu Asp Asp Leu Leu Phe Ser Ala Val Ile Glu Pro Asp Thr AlaArg Leu Asp Asp Leu Leu Phe Ser Ala Val Ile Glu Pro Asp Thr Ala

35 40 45 35 40 45

Ile Gln Glu His Gln Thr Phe Cys Gln Leu Leu Gln Glu Gln Asn IleIle Gln Glu His Gln Thr Phe Cys Gln Leu Leu Gln Glu Gln Asn Ile

50 55 60 50 55 60

Glu Val Val Gln Leu Thr Asp Leu Thr Ala Thr Thr Phe Asp Lys AlaGlu Val Val Gln Leu Thr Asp Leu Thr Ala Thr Thr Phe Asp Lys Ala

65 70 75 8065 70 75 80

Asn Ala Thr Ala Gln Asn Gln Phe Ile Glu Thr Trp Leu Asp Gln AlaAsn Ala Thr Ala Gln Asn Gln Phe Ile Glu Thr Trp Leu Asp Gln Ala

85 90 95 85 90 95

Glu Pro Lys Leu Thr Pro Glu His Arg Lys Val Ala Lys Gln Tyr LeuGlu Pro Lys Leu Thr Pro Glu His Arg Lys Val Ala Lys Gln Tyr Leu

100 105 110 100 105 110

Leu Glu Gln Lys Ala Lys Ser Thr Leu Ser Met Val Arg Ser Met MetLeu Glu Gln Lys Ala Lys Ser Thr Leu Ser Met Val Arg Ser Met Met

115 120 125 115 120 125

Gly Gly Ile Asp Lys Arg Lys Val Ala Ala Ala Asn Thr Ile Asn GlyGly Gly Ile Asp Lys Arg Lys Val Ala Ala Ala Asn Thr Ile Asn Gly

130 135 140 130 135 140

Asp Phe Leu Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp ProAsp Phe Leu Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro

145 150 155 160145 150 155 160

Phe Ala Ser Ile Gly His Gly Ile Ser Ile Asn Arg Met Lys Tyr LeuPhe Ala Ser Ile Gly His Gly Ile Ser Ile Asn Arg Met Lys Tyr Leu

165 170 175 165 170 175

Thr Arg Arg Arg Glu Thr Leu Phe Ala Ser Phe Ile Phe Ala Asn HisThr Arg Arg Arg Glu Thr Leu Phe Ala Ser Phe Ile Phe Ala Asn His

180 185 190 180 185 190

Pro Ile Ile Ala Ala Arg Lys Phe Tyr Phe Lys Pro Ile Asp Met GlyPro Ile Ile Ala Ala Arg Lys Phe Tyr Phe Lys Pro Ile Asp Met Gly

195 200 205 195 200 205

Thr Ile Glu Gly Gly Asp Ile Phe Val Tyr Asp Gln Gln Thr Val ValThr Ile Glu Gly Gly Asp Ile Phe Val Tyr Asp Gln Gln Thr Val Val

210 215 220 210 215 220

Met Gly Leu Ser Glu Arg Thr Thr Glu Ala Ala Ile Asn Val Leu AlaMet Gly Leu Ser Glu Arg Thr Thr Glu Ala Ala Ile Asn Val Leu Ala

225 230 235 240225 230 235 240

Lys Lys Ile Gln Gln Asp Ser Ser Thr Ser Phe Lys Arg Ile Phe ValLys Lys Ile Gln Gln Asp Ser Ser Thr Ser Phe Lys Arg Ile Phe Val

245 250 255 245 250 255

Ile Asn Val Pro Gln Leu Pro Asn Leu Met His Leu Asp Thr Trp LeuIle Asn Val Pro Gln Leu Pro Asn Leu Met His Leu Asp Thr Trp Leu

260 265 270 260 265 270

Thr Met Leu Asp Arg Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu AlaThr Met Leu Asp Arg Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ala

275 280 285 275 280 285

Val Leu Lys Ala Trp Arg Ile Asp Phe Thr Asp Pro Ala Leu Lys TrpVal Leu Lys Ala Trp Arg Ile Asp Phe Thr Asp Pro Ala Leu Lys Trp

290 295 300 290 295 300

Asn Glu Ile Ala Gly Asp Leu Ser Thr Ile Leu His Thr Ile Ile GlyAsn Glu Ile Ala Gly Asp Leu Ser Thr Ile Leu His Thr Ile Ile Gly

305 310 315 320305 310 315 320

Gln Lys Pro Met Leu Ile Pro Ile Ala Gly Ala Asp Ala Asn Gln ThrGln Lys Pro Met Leu Ile Pro Ile Ala Gly Ala Asp Ala Asn Gln Thr

325 330 335 325 330 335

Glu Ile Asp Ile Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr IleGlu Ile Asp Ile Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile

340 345 350 340 345 350

Ala Pro Ser Val Val Val Gly Tyr Ala Arg Asn Lys Leu Thr His GlnAla Pro Ser Val Val Val Gly Tyr Ala Arg Asn Lys Leu Thr His Gln

355 360 365 355 360 365

Thr Leu Glu Ala Ala Gly Val Lys Val Ile Ala Phe Lys Gly Asn GlnThr Leu Glu Ala Ala Gly Val Lys Val Ile Ala Phe Lys Gly Asn Gln

370 375 380 370 375 380

Leu Ser Leu Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu ValLeu Ser Leu Gly Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val

385 390 395 400385 390 395 400

Arg Lys Pro LeuArg Lys Pro Leu

<210> 55<210> 55

<211> 414<211> 414

<212> PRT<212> PRT

<213> 支原体属CAG:877（Mycoplasma sp. CAG:877）<213> Mycoplasma sp. CAG:877 (Mycoplasma sp. CAG:877)

<400> 55<400> 55

Met Glu Lys Ile His Val Thr Ser Glu Ile Gly Pro Leu Lys Lys ValMet Glu Lys Ile His Val Thr Ser Glu Ile Gly Pro Leu Lys Lys Val

1 5 10 151 5 10 15

Leu Leu His Arg Pro Gly Asn Glu Leu Leu Asn Leu Thr Pro Asp ThrLeu Leu His Arg Pro Gly Asn Glu Leu Leu Asn Leu Thr Pro Asp Thr

20 25 30 20 25 30

Leu Ser Arg Leu Leu Phe Asp Asp Ile Pro Tyr Leu Pro Asp Ala IleLeu Ser Arg Leu Leu Phe Asp Asp Ile Pro Tyr Leu Pro Asp Ala Ile

35 40 45 35 40 45

Lys Glu His Asp Glu Phe Ala Asp Ala Leu Arg Ala Asn Gly Val GluLys Glu His Asp Glu Phe Ala Asp Ala Leu Arg Ala Asn Gly Val Glu

50 55 60 50 55 60

Val Val Tyr Leu Glu Asn Leu Met Ala Asp Val Leu Asp Leu Ser AspVal Val Tyr Leu Glu Asn Leu Met Ala Asp Val Leu Asp Leu Ser Asp

65 70 75 8065 70 75 80

Glu Ile Arg Asp Lys Phe Ile Lys Gln Phe Ile Tyr Glu Ala Gly IleGlu Ile Arg Asp Lys Phe Ile Lys Gln Phe Ile Tyr Glu Ala Gly Ile

85 90 95 85 90 95

Arg Thr Pro Lys Tyr Lys Tyr Leu Val Phe Asp Tyr Leu Asp Gln IleArg Thr Pro Lys Tyr Lys Tyr Leu Val Phe Asp Tyr Leu Asp Gln Ile

100 105 110 100 105 110

Thr Asn Ser Lys Lys Leu Val Leu Lys Thr Met Glu Gly Ile Gln IleThr Asn Ser Lys Lys Lys Leu Val Leu Lys Thr Met Glu Gly Ile Gln Ile

115 120 125 115 120 125

Ser Asp Ile Pro Arg Arg Lys Arg Glu Ile Glu Lys Ser Leu Val AspSer Asp Ile Pro Arg Arg Lys Arg Glu Ile Glu Lys Ser Leu Val Asp

130 135 140 130 135 140

Leu Ile Glu Thr Glu Asp Glu Phe Ile Ala Asp Pro Met Pro Asn LeuLeu Ile Glu Thr Glu Asp Glu Phe Ile Ala Asp Pro Met Pro Asn Leu

145 150 155 160145 150 155 160

Tyr Phe Thr Arg Asp Pro Phe Ala Ser Val Gly Glu Gly Ile Ser LeuTyr Phe Thr Arg Asp Pro Phe Ala Ser Val Gly Glu Gly Ile Ser Leu

165 170 175 165 170 175

Asn Lys Met Tyr Ser Val Thr Arg Asn Arg Glu Thr Ile Tyr Ala GluAsn Lys Met Tyr Ser Val Thr Arg Asn Arg Glu Thr Ile Tyr Ala Glu

180 185 190 180 185 190

Tyr Ile Phe Lys Tyr His Pro Asp Tyr Lys Asp Gln Ala Arg Leu TyrTyr Ile Phe Lys Tyr His Pro Asp Tyr Lys Asp Gln Ala Arg Leu Tyr

195 200 205 195 200 205

Tyr Asp Arg Tyr Asn Pro Tyr His Ile Glu Gly Gly Asp Val Leu AsnTyr Asp Arg Tyr Asn Pro Tyr His Ile Glu Gly Gly Asp Val Leu Asn

210 215 220 210 215 220

Ile Asn Asp His Val Leu Ala Ile Gly Ile Ser Gln Arg Thr Thr AlaIle Asn Asp His Val Leu Ala Ile Gly Ile Ser Gln Arg Thr Thr Ala

225 230 235 240225 230 235 240

Glu Ala Ile Asp Gln Ile Ala Lys Asn Leu Phe Lys Asp Pro Glu CysGlu Ala Ile Asp Gln Ile Ala Lys Asn Leu Phe Lys Asp Pro Glu Cys

245 250 255 245 250 255

Lys Ile Asp Thr Ile Leu Ala Phe Asn Ile Pro Glu Ser Arg Ala PheLys Ile Asp Thr Ile Leu Ala Phe Asn Ile Pro Glu Ser Arg Ala Phe

260 265 270 260 265 270

Met His Leu Asp Thr Val Phe Thr Gln Val Asp Tyr Asp Lys Phe ThrMet His Leu Asp Thr Val Phe Thr Gln Val Asp Tyr Asp Lys Phe Thr

275 280 285 275 280 285

Tyr His Pro Gly Ile Met Gly Thr Leu Gln Val Phe Glu Ile Thr GluTyr His Pro Gly Ile Met Gly Thr Leu Gln Val Phe Glu Ile Thr Glu

290 295 300 290 295 300

Gly Asp Asp Pro Asn Ser Asp Glu Asp Leu Thr Val Thr Glu Ile AsnGly Asp Asp Pro Asn Ser Asp Glu Asp Leu Thr Val Thr Glu Ile Asn

305 310 315 320305 310 315 320

Ala Pro Leu Glu Glu Ile Leu Thr Lys Tyr Val Gly Arg Lys Val ThrAla Pro Leu Glu Glu Ile Leu Thr Lys Tyr Val Gly Arg Lys Val Thr

325 330 335 325 330 335

Leu Ile Pro Cys Ala Gly Gly Asp Lys Val Ser Ala Glu Arg Glu GlnLeu Ile Pro Cys Ala Gly Gly Asp Lys Val Ser Ala Glu Arg Glu Gln

340 345 350 340 345 350

Trp Asn Asp Gly Ser Asn Thr Leu Cys Ile Ala Pro Gly Val Val ValTrp Asn Asp Gly Ser Asn Thr Leu Cys Ile Ala Pro Gly Val Val Val

355 360 365 355 360 365

Val Tyr Asp Arg Asn Asn Leu Thr Asn Ala Val Leu Arg Ser Tyr GlyVal Tyr Asp Arg Asn Asn Leu Thr Asn Ala Val Leu Arg Ser Tyr Gly

370 375 380 370 375 380

Leu Lys Val Ile Glu Ile His Gly Ala Glu Leu Ser Arg Gly Arg GlyLeu Lys Val Ile Glu Ile His Gly Ala Glu Leu Ser Arg Gly Arg Gly

385 390 395 400385 390 395 400

Gly Pro Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp IleGly Pro Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp Ile

405 410 405 410

<210> 56<210> 56

<211> 408<211> 408

<212> PRT<212> PRT

<213> 支原体属CAG:472（Mycoplasma sp. CAG:472）<213> Mycoplasma sp. CAG:472 (Mycoplasma sp. CAG:472)

<400> 56<400> 56

Met His Val Thr Ser Glu Ile Lys Lys Leu Lys Lys Val Leu Val HisMet His Val Thr Ser Glu Ile Lys Lys Leu Lys Lys Val Leu Val His

1 5 10 151 5 10 15

Arg Pro Gly Lys Glu Leu Leu Asn Leu Thr Pro Asp Thr Leu Gly ArgArg Pro Gly Lys Glu Leu Leu Asn Leu Thr Pro Asp Thr Leu Gly Arg

20 25 30 20 25 30

Leu Leu Phe Asp Asp Ile Pro Tyr Leu Lys Asp Ala Ile Leu Glu HisLeu Leu Phe Asp Asp Ile Pro Tyr Leu Lys Asp Ala Ile Leu Glu His

35 40 45 35 40 45

Asp Glu Phe Cys Gln Ile Leu Arg Asp Asn Asp Val Glu Val Val TyrAsp Glu Phe Cys Gln Ile Leu Arg Asp Asn Asp Val Glu Val Val Tyr

50 55 60 50 55 60

Leu Glu Asp Leu Met Ala Glu Thr Leu Asp Glu Asn Pro Gln Val LysLeu Glu Asp Leu Met Ala Glu Thr Leu Asp Glu Asn Pro Gln Val Lys

65 70 75 8065 70 75 80

Pro Ser Phe Ile Arg Gln Phe Ile Tyr Glu Ala Gly Val Arg Thr ProPro Ser Phe Ile Arg Gln Phe Ile Tyr Glu Ala Gly Val Arg Thr Pro

85 90 95 85 90 95

Lys Tyr Lys Asp Leu Leu Phe Asp Tyr Leu Met Ser Tyr Thr Asn AsnLys Tyr Lys Asp Leu Leu Phe Asp Tyr Leu Met Ser Tyr Thr Asn Asn

100 105 110 100 105 110

Lys Glu Leu Val Leu Lys Thr Met Glu Gly Ile Lys Val Ser Glu ValLys Glu Leu Val Leu Lys Thr Met Glu Gly Ile Lys Val Ser Glu Val

115 120 125 115 120 125

His Arg Asn Lys Gln Asp Ser Glu Tyr Ser Leu Val Asp Gln Ile SerHis Arg Asn Lys Gln Asp Ser Glu Tyr Ser Leu Val Asp Gln Ile Ser

130 135 140 130 135 140

Glu Glu Thr Lys Phe Leu Ala Glu Pro Met Pro Asn Leu Tyr Phe ThrGlu Glu Thr Lys Phe Leu Ala Glu Pro Met Pro Asn Leu Tyr Phe Thr

145 150 155 160145 150 155 160

Arg Asp Pro Phe Ala Ser Val Gly Asp Gly Ile Ile Leu Asn Lys MetArg Asp Pro Phe Ala Ser Val Gly Asp Gly Ile Ile Leu Asn Lys Met

165 170 175 165 170 175

His Ser Val Thr Arg Ser Arg Glu Thr Ile Tyr Ala Tyr Tyr Ile PheHis Ser Val Thr Arg Ser Arg Glu Thr Ile Tyr Ala Tyr Tyr Ile Phe

180 185 190 180 185 190

Asn Tyr His Pro Asp Tyr Met Asp Lys Val Pro Lys Tyr Tyr Asp ArgAsn Tyr His Pro Asp Tyr Met Asp Lys Val Pro Lys Tyr Tyr Asp Arg

195 200 205 195 200 205

Glu Asn Pro Phe Ser Ile Glu Gly Gly Asp Val Leu Asn Leu Asn GluGlu Asn Pro Phe Ser Ile Glu Gly Gly Asp Val Leu Asn Leu Asn Glu

210 215 220 210 215 220

His Thr Leu Ala Ile Gly Ile Ser Gln Arg Thr Ser Ala Glu Ala IleHis Thr Leu Ala Ile Gly Ile Ser Gln Arg Thr Ser Ala Glu Ala Ile

225 230 235 240225 230 235 240

Asp Leu Val Ala Lys Asn Met Phe Asn Asp Glu Lys Cys Asn Ile AspAsp Leu Val Ala Lys Asn Met Phe Asn Asp Glu Lys Cys Asn Ile Asp

245 250 255 245 250 255

Thr Ile Leu Ala Phe Lys Ile Pro Glu Cys Arg Ala Phe Met His LeuThr Ile Leu Ala Phe Lys Ile Pro Glu Cys Arg Ala Phe Met His Leu

260 265 270 260 265 270

Asp Thr Val Phe Thr Gln Ile Asp Ile Asp Lys Phe Thr Tyr His ProAsp Thr Val Phe Thr Gln Ile Asp Ile Asp Lys Phe Thr Tyr His Pro

275 280 285 275 280 285

Gly Ile Met Asp Thr Leu Glu Val Phe Glu Ile Thr Lys Asn Glu AspGly Ile Met Asp Thr Leu Glu Val Phe Glu Ile Thr Lys Asn Glu Asp

290 295 300 290 295 300

Asp Leu Asp Glu Val Arg Val Ile Lys Lys Glu Gly Ser Leu Glu AsnAsp Leu Asp Glu Val Arg Val Ile Lys Lys Glu Gly Ser Leu Glu Asn

305 310 315 320305 310 315 320

Ile Leu Glu Glu Tyr Leu Gly Ile Asp Ile Thr Leu Ile Pro Cys AlaIle Leu Glu Glu Tyr Leu Gly Ile Asp Ile Thr Leu Ile Pro Cys Ala

325 330 335 325 330 335

Gly Gly Asp Lys Ile Ala Ser Glu Arg Glu Gln Trp Asn Asp Gly ThrGly Gly Asp Lys Ile Ala Ser Glu Arg Glu Gln Trp Asn Asp Gly Thr

340 345 350 340 345 350

Asn Thr Leu Cys Ile Ala Pro Gly Val Val Val Val Tyr Asn Arg AsnAsn Thr Leu Cys Ile Ala Pro Gly Val Val Val Val Tyr Asn Arg Asn

355 360 365 355 360 365

Asn Ile Thr Asn Glu Val Leu Arg Glu Lys Gly Ile Lys Val Ile GluAsn Ile Thr Asn Glu Val Leu Arg Glu Lys Gly Ile Lys Val Ile Glu

370 375 380 370 375 380

Met Asn Ser Ala Glu Leu Ser Arg Gly Arg Gly Gly Pro Arg Cys MetMet Asn Ser Ala Glu Leu Ser Arg Gly Arg Gly Gly Pro Arg Cys Met

385 390 395 400385 390 395 400

Ser Met Pro Leu Glu Arg Glu AspSer Met Pro Leu Glu Arg Glu Asp

405 405

<210> 57<210> 57

<211> 401<211> 401

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 鸽支原体-鸡支原体嵌合ADI<223> Laboratory Preparation - M. pigeonii-M. gallis chimeric ADI

<400> 57<400> 57

Met Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Glu Leu Lys Glu ValMet Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Glu Leu Lys Glu Val

1 5 10 151 5 10 15

Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser ArgLeu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser Arg

20 25 30 20 25 30

Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Asn Glu Ala IleLeu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Asn Glu Ala Ile

35 40 45 35 40 45

Lys Glu His Lys Gly Phe Leu Lys Ile Leu Gln Asp Lys Gly Ile LysLys Glu His Lys Gly Phe Leu Lys Ile Leu Gln Asp Lys Gly Ile Lys

50 55 60 50 55 60

Val Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Val Lys Tyr AlaVal Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Val Lys Tyr Ala

65 70 75 8065 70 75 80

Thr Ala Glu Gln Lys Ala Ala Phe Ile Glu Lys Tyr Leu Asp Glu AlaThr Ala Glu Gln Lys Ala Ala Phe Ile Glu Lys Tyr Leu Asp Glu Ala

85 90 95 85 90 95

Thr Pro Ala Leu Ser Ala Glu Asn Arg Glu Arg Ala Lys Lys Tyr IleThr Pro Ala Leu Ser Ala Glu Asn Arg Glu Arg Ala Lys Lys Tyr Ile

100 105 110 100 105 110

Leu Ser Leu Glu Met Gln Pro Val Lys Met Ile Arg Thr Met Met AlaLeu Ser Leu Glu Met Gln Pro Val Lys Met Ile Arg Thr Met Met Ala

115 120 125 115 120 125

Gly Leu Ser Lys Tyr Glu Leu Asn Val Glu Ser Asn Ile Glu Leu IleGly Leu Ser Lys Tyr Glu Leu Asn Val Glu Ser Asn Ile Glu Leu Ile

130 135 140 130 135 140

Ile Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala SerIle Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser

145 150 155 160145 150 155 160

Ala Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Thr Arg LysAla Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Thr Arg Lys

165 170 175 165 170 175

Arg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ala Thr His Pro Asp TyrArg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ala Thr His Pro Asp Tyr

180 185 190 180 185 190

Lys Thr Thr Pro His Trp Phe Asp Arg Leu Asp Glu Gly Asn Ile GluLys Thr Thr Pro His Trp Phe Asp Arg Leu Asp Glu Gly Asn Ile Glu

195 200 205 195 200 205

Gly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly ValGly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly Val

210 215 220 210 215 220

Ser Glu Arg Thr Asn Lys Glu Ala Ile Leu Thr Ile Ala Lys Lys IleSer Glu Arg Thr Asn Lys Glu Ala Ile Leu Thr Ile Ala Lys Lys Ile

225 230 235 240225 230 235 240

Lys Asn Asn Lys Glu Ala Lys Phe Lys Lys Ile Val Ala Ile Asn ValLys Asn Asn Lys Glu Ala Lys Phe Lys Lys Ile Val Ala Ile Asn Val

245 250 255 245 250 255

Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met ValPro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Val

260 265 270 260 265 270

Asp Lys Asp Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu LysAsp Lys Asp Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu Lys

275 280 285 275 280 285

Val Trp Glu Ile Asp Leu Ser Lys Glu Ile Glu Met Val Glu Thr AsnVal Trp Glu Ile Asp Leu Ser Lys Glu Ile Glu Met Val Glu Thr Asn

290 295 300 290 295 300

Lys Pro Leu Ala Asp Val Leu Glu Ser Ile Ile Gly Val Lys Pro ValLys Pro Leu Ala Asp Val Leu Glu Ser Ile Ile Gly Val Lys Pro Val

305 310 315 320305 310 315 320

Leu Ile Pro Ile Ala Gly Lys Gly Ala Thr Gln Leu Asp Ile Asp IleLeu Ile Pro Ile Ala Gly Lys Gly Ala Thr Gln Leu Asp Ile Asp Ile

325 330 335 325 330 335

Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly ValGlu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly Val

340 345 350 340 345 350

Val Val Gly Tyr Ser Arg Asn Ile Lys Thr Glu Ala Ala Leu Arg AlaVal Val Gly Tyr Ser Arg Asn Ile Lys Thr Glu Ala Ala Leu Arg Ala

355 360 365 355 360 365

Ala Gly Val Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu GlyAla Gly Val Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu Gly

370 375 380 370 375 380

Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp ValMet Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp Val

385 390 395 400385 390 395 400

LysLys

<210> 58<210> 58

<211> 401<211> 401

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 鸽支原体-惰性支原体嵌合ADI<223> Laboratory Preparation - M. pigeonii-M. inert chimeric ADI

<400> 58<400> 58

Met Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Glu Leu Lys Glu ValMet Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Glu Leu Lys Glu Val

1 5 10 151 5 10 15

Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser ArgLeu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser Arg

20 25 30 20 25 30

Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Asn Glu Ala IleLeu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Asn Glu Ala Ile

35 40 45 35 40 45

Lys Glu His Lys Gly Phe Leu Lys Ile Leu Gln Asp Lys Gly Ile LysLys Glu His Lys Gly Phe Leu Lys Ile Leu Gln Asp Lys Gly Ile Lys

50 55 60 50 55 60

Val Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Lys His Tyr AlaVal Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Lys His Tyr Ala

65 70 75 8065 70 75 80

Ser Glu Ala Glu Lys Glu Ala Phe Ile Glu Lys Tyr Leu Asp Glu AlaSer Glu Ala Glu Lys Glu Ala Phe Ile Glu Lys Tyr Leu Asp Glu Ala

85 90 95 85 90 95

Thr Pro Val Leu Ser Lys Asp Met Arg Ala Lys Val Lys Asn Tyr IleThr Pro Val Leu Ser Lys Asp Met Arg Ala Lys Val Lys Asn Tyr Ile

100 105 110 100 105 110

Leu Ser Met Gln Gly Glu Pro Val Lys Met Val Arg Thr Met Met AlaLeu Ser Met Gln Gly Glu Pro Val Lys Met Val Arg Thr Met Met Ala

115 120 125 115 120 125

Gly Val Ser Lys Gln Glu Leu Asn Val Glu Ser Glu Val Glu Leu IleGly Val Ser Lys Gln Glu Leu Asn Val Glu Ser Glu Val Glu Leu Ile

130 135 140 130 135 140

Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala SerVal Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser

145 150 155 160145 150 155 160

Ala Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Thr Arg LysAla Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Thr Arg Lys

165 170 175 165 170 175

Arg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ala Thr His Pro Asp TyrArg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ala Thr His Pro Asp Tyr

180 185 190 180 185 190

Lys Thr Thr Pro His Trp Phe Asp Arg Leu Asp Glu Gly Asn Ile GluLys Thr Thr Pro His Trp Phe Asp Arg Leu Asp Glu Gly Asn Ile Glu

195 200 205 195 200 205

Gly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly ValGly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly Val

210 215 220 210 215 220

Ser Glu Arg Thr Asn Lys Glu Ala Ile Leu Thr Ile Ala Lys Lys IleSer Glu Arg Thr Asn Lys Glu Ala Ile Leu Thr Ile Ala Lys Lys Ile

225 230 235 240225 230 235 240

Lys Asn Asn Lys Glu Ala Lys Phe Lys Lys Ile Val Ala Ile Asn ValLys Asn Asn Lys Glu Ala Lys Phe Lys Lys Ile Val Ala Ile Asn Val

245 250 255 245 250 255

Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met ValPro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Val

260 265 270 260 265 270

Asp Lys Asp Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu LysAsp Lys Asp Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu Lys

275 280 285 275 280 285

Val Trp Glu Ile Asp Leu Ser Lys Glu Ile Glu Met Val Glu Thr AsnVal Trp Glu Ile Asp Leu Ser Lys Glu Ile Glu Met Val Glu Thr Asn

290 295 300 290 295 300

Lys Pro Leu Ala Asp Val Leu Glu Ser Ile Ile Gly Val Lys Pro ValLys Pro Leu Ala Asp Val Leu Glu Ser Ile Ile Gly Val Lys Pro Val

305 310 315 320305 310 315 320

Leu Ile Pro Ile Ala Gly Lys Gly Ala Thr Gln Leu Asp Ile Asp IleLeu Ile Pro Ile Ala Gly Lys Gly Ala Thr Gln Leu Asp Ile Asp Ile

325 330 335 325 330 335

Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly ValGlu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly Val

340 345 350 340 345 350

Val Val Gly Tyr Ser Arg Asn Ile Lys Thr Glu Ala Ala Leu Arg AlaVal Val Gly Tyr Ser Arg Asn Ile Lys Thr Glu Ala Ala Leu Arg Ala

355 360 365 355 360 365

Ala Gly Val Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu GlyAla Gly Val Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu Gly

370 375 380 370 375 380

Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp ValMet Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp Val

385 390 395 400385 390 395 400

LysLys

<210> 59<210> 59

<211> 401<211> 401

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 鸽支原体-火鸡支原体嵌合ADI<223> Laboratory Preparation - M. pigeon-M. turkey chimeric ADI

<400> 59<400> 59

Met Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Glu Leu Lys Glu ValMet Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Glu Leu Lys Glu Val

1 5 10 151 5 10 15

Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser ArgLeu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser Arg

20 25 30 20 25 30

Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Asn Glu Ala IleLeu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Asn Glu Ala Ile

35 40 45 35 40 45

Lys Glu His Lys Gly Phe Leu Lys Ile Leu Gln Asp Lys Gly Ile LysLys Glu His Lys Gly Phe Leu Lys Ile Leu Gln Asp Lys Gly Ile Lys

50 55 60 50 55 60

Val Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Val Lys Tyr AlaVal Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Val Lys Tyr Ala

65 70 75 8065 70 75 80

Thr Ser Lys Glu Lys Glu Ser Phe Ile Glu Lys Trp Leu Asp Glu AlaThr Ser Lys Glu Lys Glu Ser Phe Ile Glu Lys Trp Leu Asp Glu Ala

85 90 95 85 90 95

Thr Pro Ala Leu Asn Ser Glu Asn Arg Ala Arg Val Lys Asn Tyr IleThr Pro Ala Leu Asn Ser Glu Asn Arg Ala Arg Val Lys Asn Tyr Ile

100 105 110 100 105 110

Thr Ala Met Gln Gly Gln Pro Val Lys Met Val Arg Ala Met Met AlaThr Ala Met Gln Gly Gln Pro Val Lys Met Val Arg Ala Met Met Ala

115 120 125 115 120 125

Gly Val Ser Lys Gln Glu Leu Asn Ile Glu Ser Asp Val Glu Leu IleGly Val Ser Lys Gln Glu Leu Asn Ile Glu Ser Asp Val Glu Leu Ile

130 135 140 130 135 140

Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala SerVal Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser

145 150 155 160145 150 155 160

Ala Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Thr Arg LysAla Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Thr Arg Lys

165 170 175 165 170 175

Arg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ala Thr His Pro Asp TyrArg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ala Thr His Pro Asp Tyr

180 185 190 180 185 190

Lys Thr Thr Pro His Trp Phe Asp Arg Leu Asp Glu Gly Asn Ile GluLys Thr Thr Pro His Trp Phe Asp Arg Leu Asp Glu Gly Asn Ile Glu

195 200 205 195 200 205

Gly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly ValGly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly Val

210 215 220 210 215 220

Ser Glu Arg Thr Asn Lys Glu Ala Ile Leu Thr Ile Ala Lys Lys IleSer Glu Arg Thr Asn Lys Glu Ala Ile Leu Thr Ile Ala Lys Lys Ile

225 230 235 240225 230 235 240

Lys Asn Asn Lys Glu Ala Lys Phe Lys Lys Ile Val Ala Ile Asn ValLys Asn Asn Lys Glu Ala Lys Phe Lys Lys Ile Val Ala Ile Asn Val

245 250 255 245 250 255

Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met ValPro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Val

260 265 270 260 265 270

Asp Lys Asp Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu LysAsp Lys Asp Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu Lys

275 280 285 275 280 285

Val Trp Glu Ile Asp Leu Ser Lys Glu Ile Glu Met Val Glu Thr AsnVal Trp Glu Ile Asp Leu Ser Lys Glu Ile Glu Met Val Glu Thr Asn

290 295 300 290 295 300

Lys Pro Leu Ala Asp Val Leu Glu Ser Ile Ile Gly Val Lys Pro ValLys Pro Leu Ala Asp Val Leu Glu Ser Ile Ile Gly Val Lys Pro Val

305 310 315 320305 310 315 320

Leu Ile Pro Ile Ala Gly Lys Gly Ala Thr Gln Leu Asp Ile Asp IleLeu Ile Pro Ile Ala Gly Lys Gly Ala Thr Gln Leu Asp Ile Asp Ile

325 330 335 325 330 335

Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly ValGlu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly Val

340 345 350 340 345 350

Val Val Gly Tyr Ser Arg Asn Ile Lys Thr Glu Ala Ala Leu Arg AlaVal Val Gly Tyr Ser Arg Asn Ile Lys Thr Glu Ala Ala Leu Arg Ala

355 360 365 355 360 365

Ala Gly Val Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu GlyAla Gly Val Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu Gly

370 375 380 370 375 380

Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp ValMet Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp Val

385 390 395 400385 390 395 400

LysLys

<210> 60<210> 60

<211> 401<211> 401

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 鸡支原体-鸽支原体嵌合ADI<223> Laboratory Preparation - M. gallis-M. pigeon chimeric ADI

<400> 60<400> 60

Met Ser Lys Ile Arg Val Tyr Ser Glu Ile Gly Asn Leu Lys Lys ValMet Ser Lys Ile Arg Val Tyr Ser Glu Ile Gly Asn Leu Lys Lys Val

1 5 10 151 5 10 15

Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser ArgLeu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser Arg

20 25 30 20 25 30

Leu Glu Glu Leu Leu Phe Ser Ala Val Leu Glu Pro Asn Ala Ala IleLeu Glu Glu Leu Leu Phe Ser Ala Val Leu Glu Pro Asn Ala Ala Ile

35 40 45 35 40 45

Glu Glu His Lys Arg Phe Val Lys Leu Leu Glu Asp Arg Gly Ile GlnGlu Glu His Lys Arg Phe Val Lys Leu Leu Glu Asp Arg Gly Ile Gln

50 55 60 50 55 60

Ala Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Thr Tyr His AlaAla Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Thr Tyr His Ala

65 70 75 8065 70 75 80

Thr Gln Lys Glu Arg Glu Ala Phe Ile Glu Lys Trp Leu Asp Glu AlaThr Gln Lys Glu Arg Glu Ala Phe Ile Glu Lys Trp Leu Asp Glu Ala

85 90 95 85 90 95

Glu Pro Ala Leu Thr Lys Asp Leu Arg Ala Lys Val Lys Ser Tyr ValGlu Pro Ala Leu Thr Lys Asp Leu Arg Ala Lys Val Lys Ser Tyr Val

100 105 110 100 105 110

Leu Ser Lys Glu Gly Thr Pro Val Ala Met Val Arg Thr Met Met AlaLeu Ser Lys Glu Gly Thr Pro Val Ala Met Val Arg Thr Met Met Ala

115 120 125 115 120 125

Gly Val Ser Lys Gln Glu Leu Asn Val Glu Ser Glu Thr Glu Leu ValGly Val Ser Lys Gln Glu Leu Asn Val Glu Ser Glu Thr Glu Leu Val

130 135 140 130 135 140

Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala SerVal Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser

145 150 155 160145 150 155 160

Ala Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg LysAla Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg Lys

165 170 175 165 170 175

Arg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ala Ile His Pro Glu TyrArg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ala Ile His Pro Glu Tyr

180 185 190 180 185 190

Lys Glu Thr Pro His Trp Phe Asp Arg Leu Asp His Gly Ser Ile GluLys Glu Thr Pro His Trp Phe Asp Arg Leu Asp His Gly Ser Ile Glu

195 200 205 195 200 205

Gly Gly Asp Val Phe Val Tyr Asn Lys Asp Thr Leu Val Ile Gly ValGly Gly Asp Val Phe Val Tyr Asn Lys Asp Thr Leu Val Ile Gly Val

210 215 220 210 215 220

Ser Glu Arg Thr Asn Lys Glu Ala Ile Ile Thr Ile Ala Lys His IleSer Glu Arg Thr Asn Lys Glu Ala Ile Ile Thr Ile Ala Lys His Ile

225 230 235 240225 230 235 240

Gln Asp Asn Lys Glu Ala Glu Phe Lys Lys Ile Val Ala Ile Asn ValGln Asp Asn Lys Glu Ala Glu Phe Lys Lys Ile Val Ala Ile Asn Val

245 250 255 245 250 255

Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met ValPro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Val

260 265 270 260 265 270

Asp Lys Asn Lys Phe Ile Tyr Ser Pro Asn Met Leu Ser Val Leu LysAsp Lys Asn Lys Phe Ile Tyr Ser Pro Asn Met Leu Ser Val Leu Lys

275 280 285 275 280 285

Ile Trp Glu Ile Asp Leu Ala Lys Pro Ile Glu Met Val Glu Ser AsnIle Trp Glu Ile Asp Leu Ala Lys Pro Ile Glu Met Val Glu Ser Asn

290 295 300 290 295 300

Lys Ser Leu Thr Glu Val Leu Glu Ser Ile Ile Gly Glu Lys Pro IleLys Ser Leu Thr Glu Val Leu Glu Ser Ile Ile Gly Glu Lys Pro Ile

305 310 315 320305 310 315 320

Leu Ile Pro Ile Ala Gly Glu Gly Ala Ser Gln Leu Asp Ile Asp IleLeu Ile Pro Ile Ala Gly Glu Gly Ala Ser Gln Leu Asp Ile Asp Ile

325 330 335 325 330 335

Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly ValGlu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly Val

340 345 350 340 345 350

Val Val Gly Tyr Ser Arg Asn Glu Lys Thr Glu Lys Ala Leu Lys AlaVal Val Gly Tyr Ser Arg Asn Glu Lys Thr Glu Lys Ala Leu Lys Ala

355 360 365 355 360 365

Ala Gly Ile Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu GlyAla Gly Ile Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu Gly

370 375 380 370 375 380

Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp ValMet Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp Val

385 390 395 400385 390 395 400

LysLys

<210> 61<210> 61

<211> 401<211> 401

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 鸡支原体-惰性支原体嵌合ADI<223> Laboratory Preparation - M. gallinarum-M. inert chimeric ADI

<400> 61<400> 61

Met Ser Lys Ile Arg Val Tyr Ser Glu Ile Gly Asn Leu Lys Lys ValMet Ser Lys Ile Arg Val Tyr Ser Glu Ile Gly Asn Leu Lys Lys Val

1 5 10 151 5 10 15

Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser ArgLeu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser Arg

20 25 30 20 25 30

Leu Glu Glu Leu Leu Phe Ser Ala Val Leu Glu Pro Asn Ala Ala IleLeu Glu Glu Leu Leu Phe Ser Ala Val Leu Glu Pro Asn Ala Ala Ile

35 40 45 35 40 45

Glu Glu His Lys Arg Phe Val Lys Leu Leu Glu Asp Arg Gly Ile GlnGlu Glu His Lys Arg Phe Val Lys Leu Leu Glu Asp Arg Gly Ile Gln

50 55 60 50 55 60

Ala Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Lys His Tyr AlaAla Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Lys His Tyr Ala

65 70 75 8065 70 75 80

Ser Glu Ala Glu Lys Glu Ala Phe Ile Glu Lys Tyr Leu Asp Glu AlaSer Glu Ala Glu Lys Glu Ala Phe Ile Glu Lys Tyr Leu Asp Glu Ala

85 90 95 85 90 95

Thr Pro Val Leu Ser Lys Asp Met Arg Ala Lys Val Lys Asn Tyr IleThr Pro Val Leu Ser Lys Asp Met Arg Ala Lys Val Lys Asn Tyr Ile

100 105 110 100 105 110

Leu Ser Met Gln Gly Glu Pro Val Lys Met Val Arg Thr Met Met AlaLeu Ser Met Gln Gly Glu Pro Val Lys Met Val Arg Thr Met Met Ala

115 120 125 115 120 125

Gly Val Ser Lys Gln Glu Leu Asn Val Glu Ser Glu Val Glu Leu IleGly Val Ser Lys Gln Glu Leu Asn Val Glu Ser Glu Val Glu Leu Ile

130 135 140 130 135 140

Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala SerVal Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser

145 150 155 160145 150 155 160

Ala Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg LysAla Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg Lys

165 170 175 165 170 175

Arg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ala Ile His Pro Glu TyrArg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ala Ile His Pro Glu Tyr

180 185 190 180 185 190

Lys Glu Thr Pro His Trp Phe Asp Arg Leu Asp His Gly Ser Ile GluLys Glu Thr Pro His Trp Phe Asp Arg Leu Asp His Gly Ser Ile Glu

195 200 205 195 200 205

Gly Gly Asp Val Phe Val Tyr Asn Lys Asp Thr Leu Val Ile Gly ValGly Gly Asp Val Phe Val Tyr Asn Lys Asp Thr Leu Val Ile Gly Val

210 215 220 210 215 220

Ser Glu Arg Thr Asn Lys Glu Ala Ile Ile Thr Ile Ala Lys His IleSer Glu Arg Thr Asn Lys Glu Ala Ile Ile Thr Ile Ala Lys His Ile

225 230 235 240225 230 235 240

Gln Asp Asn Lys Glu Ala Glu Phe Lys Lys Ile Val Ala Ile Asn ValGln Asp Asn Lys Glu Ala Glu Phe Lys Lys Ile Val Ala Ile Asn Val

245 250 255 245 250 255

Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met ValPro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Val

260 265 270 260 265 270

Asp Lys Asn Lys Phe Ile Tyr Ser Pro Asn Met Leu Ser Val Leu LysAsp Lys Asn Lys Phe Ile Tyr Ser Pro Asn Met Leu Ser Val Leu Lys

275 280 285 275 280 285

Ile Trp Glu Ile Asp Leu Ala Lys Pro Ile Glu Met Val Glu Ser AsnIle Trp Glu Ile Asp Leu Ala Lys Pro Ile Glu Met Val Glu Ser Asn

290 295 300 290 295 300

Lys Ser Leu Thr Glu Val Leu Glu Ser Ile Ile Gly Glu Lys Pro IleLys Ser Leu Thr Glu Val Leu Glu Ser Ile Ile Gly Glu Lys Pro Ile

305 310 315 320305 310 315 320

Leu Ile Pro Ile Ala Gly Glu Gly Ala Ser Gln Leu Asp Ile Asp IleLeu Ile Pro Ile Ala Gly Glu Gly Ala Ser Gln Leu Asp Ile Asp Ile

325 330 335 325 330 335

Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly ValGlu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly Val

340 345 350 340 345 350

Val Val Gly Tyr Ser Arg Asn Glu Lys Thr Glu Lys Ala Leu Lys AlaVal Val Gly Tyr Ser Arg Asn Glu Lys Thr Glu Lys Ala Leu Lys Ala

355 360 365 355 360 365

Ala Gly Ile Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu GlyAla Gly Ile Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu Gly

370 375 380 370 375 380

Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp ValMet Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp Val

385 390 395 400385 390 395 400

LysLys

<210> 62<210> 62

<211> 401<211> 401

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 鸡支原体-火鸡支原体嵌合ADI<223> Laboratory Preparation - M. gallis-M. turkeyi Chimeric ADI

<400> 62<400> 62

Met Ser Lys Ile Arg Val Tyr Ser Glu Ile Gly Asn Leu Lys Lys ValMet Ser Lys Ile Arg Val Tyr Ser Glu Ile Gly Asn Leu Lys Lys Val

1 5 10 151 5 10 15

Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser ArgLeu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser Arg

20 25 30 20 25 30

Leu Glu Glu Leu Leu Phe Ser Ala Val Leu Glu Pro Asn Ala Ala IleLeu Glu Glu Leu Leu Phe Ser Ala Val Leu Glu Pro Asn Ala Ala Ile

35 40 45 35 40 45

Glu Glu His Lys Arg Phe Val Lys Leu Leu Glu Asp Arg Gly Ile GlnGlu Glu His Lys Arg Phe Val Lys Leu Leu Glu Asp Arg Gly Ile Gln

50 55 60 50 55 60

Ala Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Val Lys Tyr AlaAla Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Val Lys Tyr Ala

65 70 75 8065 70 75 80

Thr Ser Lys Glu Lys Glu Ser Phe Ile Glu Lys Trp Leu Asp Glu AlaThr Ser Lys Glu Lys Glu Ser Phe Ile Glu Lys Trp Leu Asp Glu Ala

85 90 95 85 90 95

Thr Pro Ala Leu Asn Ser Glu Asn Arg Ala Arg Val Lys Asn Tyr IleThr Pro Ala Leu Asn Ser Glu Asn Arg Ala Arg Val Lys Asn Tyr Ile

100 105 110 100 105 110

Thr Ala Met Gln Gly Gln Pro Val Lys Met Val Arg Ala Met Met AlaThr Ala Met Gln Gly Gln Pro Val Lys Met Val Arg Ala Met Met Ala

115 120 125 115 120 125

Gly Val Ser Lys Gln Glu Leu Asn Ile Glu Ser Asp Val Glu Leu IleGly Val Ser Lys Gln Glu Leu Asn Ile Glu Ser Asp Val Glu Leu Ile

130 135 140 130 135 140

Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala SerVal Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser

145 150 155 160145 150 155 160

Ala Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg LysAla Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg Lys

165 170 175 165 170 175

Arg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ala Ile His Pro Glu TyrArg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ala Ile His Pro Glu Tyr

180 185 190 180 185 190

Lys Glu Thr Pro His Trp Phe Asp Arg Leu Asp His Gly Ser Ile GluLys Glu Thr Pro His Trp Phe Asp Arg Leu Asp His Gly Ser Ile Glu

195 200 205 195 200 205

Gly Gly Asp Val Phe Val Tyr Asn Lys Asp Thr Leu Val Ile Gly ValGly Gly Asp Val Phe Val Tyr Asn Lys Asp Thr Leu Val Ile Gly Val

210 215 220 210 215 220

Ser Glu Arg Thr Asn Lys Glu Ala Ile Ile Thr Ile Ala Lys His IleSer Glu Arg Thr Asn Lys Glu Ala Ile Ile Thr Ile Ala Lys His Ile

225 230 235 240225 230 235 240

Gln Asp Asn Lys Glu Ala Glu Phe Lys Lys Ile Val Ala Ile Asn ValGln Asp Asn Lys Glu Ala Glu Phe Lys Lys Ile Val Ala Ile Asn Val

245 250 255 245 250 255

Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met ValPro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Val

260 265 270 260 265 270

Asp Lys Asn Lys Phe Ile Tyr Ser Pro Asn Met Leu Ser Val Leu LysAsp Lys Asn Lys Phe Ile Tyr Ser Pro Asn Met Leu Ser Val Leu Lys

275 280 285 275 280 285

Ile Trp Glu Ile Asp Leu Ala Lys Pro Ile Glu Met Val Glu Ser AsnIle Trp Glu Ile Asp Leu Ala Lys Pro Ile Glu Met Val Glu Ser Asn

290 295 300 290 295 300

Lys Ser Leu Thr Glu Val Leu Glu Ser Ile Ile Gly Glu Lys Pro IleLys Ser Leu Thr Glu Val Leu Glu Ser Ile Ile Gly Glu Lys Pro Ile

305 310 315 320305 310 315 320

Leu Ile Pro Ile Ala Gly Glu Gly Ala Ser Gln Leu Asp Ile Asp IleLeu Ile Pro Ile Ala Gly Glu Gly Ala Ser Gln Leu Asp Ile Asp Ile

325 330 335 325 330 335

Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly ValGlu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly Val

340 345 350 340 345 350

Val Val Gly Tyr Ser Arg Asn Glu Lys Thr Glu Lys Ala Leu Lys AlaVal Val Gly Tyr Ser Arg Asn Glu Lys Thr Glu Lys Ala Leu Lys Ala

355 360 365 355 360 365

Ala Gly Ile Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu GlyAla Gly Ile Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu Gly

370 375 380 370 375 380

Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp ValMet Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp Val

385 390 395 400385 390 395 400

LysLys

<210> 63<210> 63

<211> 401<211> 401

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 惰性支原体-鸽支原体嵌合ADI<223> Laboratory Preparation - M. inert-M. pigeon chimeric ADI

<400> 63<400> 63

Met Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Val Leu Lys Glu ValMet Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Val Leu Lys Glu Val

1 5 10 151 5 10 15

Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ala Pro Ser ArgLeu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ala Pro Ser Arg

20 25 30 20 25 30

Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Ser Ala Ala IleLeu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Ser Ala Ala Ile

35 40 45 35 40 45

Gln Glu His Lys Ser Phe Leu Lys Ile Leu Gln Asp Arg Gly Ile LysGln Glu His Lys Ser Phe Leu Lys Ile Leu Gln Asp Arg Gly Ile Lys

50 55 60 50 55 60

Thr Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Thr Tyr His AlaThr Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Thr Tyr His Ala

65 70 75 8065 70 75 80

Thr Gln Lys Glu Arg Glu Ala Phe Ile Glu Lys Trp Leu Asp Glu AlaThr Gln Lys Glu Arg Glu Ala Phe Ile Glu Lys Trp Leu Asp Glu Ala

85 90 95 85 90 95

Glu Pro Ala Leu Thr Lys Asp Leu Arg Ala Lys Val Lys Ser Tyr ValGlu Pro Ala Leu Thr Lys Asp Leu Arg Ala Lys Val Lys Ser Tyr Val

100 105 110 100 105 110

Leu Ser Lys Glu Gly Thr Pro Val Ala Met Val Arg Thr Met Met AlaLeu Ser Lys Glu Gly Thr Pro Val Ala Met Val Arg Thr Met Met Ala

115 120 125 115 120 125

Gly Val Ser Lys Gln Glu Leu Asn Val Glu Ser Glu Thr Glu Leu ValGly Val Ser Lys Gln Glu Leu Asn Val Glu Ser Glu Thr Glu Leu Val

130 135 140 130 135 140

Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala SerVal Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser

145 150 155 160145 150 155 160

Ala Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg LysAla Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg Lys

165 170 175 165 170 175

Arg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ser Ile His Pro Glu TyrArg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ser Ile His Pro Glu Tyr

180 185 190 180 185 190

Lys Lys Thr Pro His Trp Phe Asp Arg Leu Asp Asn Gly Ser Ile GluLys Lys Thr Pro His Trp Phe Asp Arg Leu Asp Asn Gly Ser Ile Glu

195 200 205 195 200 205

Gly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly ValGly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly Val

210 215 220 210 215 220

Ser Glu Arg Thr Asn Lys Glu Ala Ile Ile Thr Ile Ala Lys His IleSer Glu Arg Thr Asn Lys Glu Ala Ile Ile Thr Ile Ala Lys His Ile

225 230 235 240225 230 235 240

Gln Asp Asn Lys Glu Ala Gln Phe Lys Lys Ile Val Ala Ile Asn ValGln Asp Asn Lys Glu Ala Gln Phe Lys Lys Ile Val Ala Ile Asn Val

245 250 255 245 250 255

Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met ValPro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Val

260 265 270 260 265 270

Asp Lys Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu LysAsp Lys Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu Lys

275 280 285 275 280 285

Val Trp Glu Ile Asp Leu Ser Lys Pro Ile Glu Met Val Glu Thr AsnVal Trp Glu Ile Asp Leu Ser Lys Pro Ile Glu Met Val Glu Thr Asn

290 295 300 290 295 300

Lys Pro Leu Ala Glu Val Leu Glu Ser Ile Ile Gly Glu Lys Pro IleLys Pro Leu Ala Glu Val Leu Glu Ser Ile Ile Gly Glu Lys Pro Ile

305 310 315 320305 310 315 320

Leu Ile Pro Ile Ala Gly Lys Asp Ala Thr Gln Leu Asp Ile Asp IleLeu Ile Pro Ile Ala Gly Lys Asp Ala Thr Gln Leu Asp Ile Asp Ile

325 330 335 325 330 335

Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly ValGlu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly Val

340 345 350 340 345 350

Val Val Gly Tyr Ser Arg Asn Val Lys Thr Glu Ala Ala Leu Arg AlaVal Val Gly Tyr Ser Arg Asn Val Lys Thr Glu Ala Ala Leu Arg Ala

355 360 365 355 360 365

Ala Gly Val Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu GlyAla Gly Val Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu Gly

370 375 380 370 375 380

Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp ValMet Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp Val

385 390 395 400385 390 395 400

LysLys

<210> 64<210> 64

<211> 401<211> 401

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 惰性支原体-鸡支原体嵌合ADI<223> Laboratory Preparation - M. inert-M. gallis chimeric ADI

<400> 64<400> 64

Met Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Val Leu Lys Glu ValMet Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Val Leu Lys Glu Val

1 5 10 151 5 10 15

Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ala Pro Ser ArgLeu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ala Pro Ser Arg

20 25 30 20 25 30

Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Ser Ala Ala IleLeu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Ser Ala Ala Ile

35 40 45 35 40 45

Gln Glu His Lys Ser Phe Leu Lys Ile Leu Gln Asp Arg Gly Ile LysGln Glu His Lys Ser Phe Leu Lys Ile Leu Gln Asp Arg Gly Ile Lys

50 55 60 50 55 60

Thr Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Val Lys Tyr AlaThr Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Val Lys Tyr Ala

65 70 75 8065 70 75 80

Thr Ala Glu Gln Lys Ala Ala Phe Ile Glu Lys Tyr Leu Asp Glu AlaThr Ala Glu Gln Lys Ala Ala Phe Ile Glu Lys Tyr Leu Asp Glu Ala

85 90 95 85 90 95

Thr Pro Ala Leu Ser Ala Glu Asn Arg Glu Arg Ala Lys Lys Tyr IleThr Pro Ala Leu Ser Ala Glu Asn Arg Glu Arg Ala Lys Lys Tyr Ile

100 105 110 100 105 110

Leu Ser Leu Glu Met Gln Pro Val Lys Met Ile Arg Thr Met Met AlaLeu Ser Leu Glu Met Gln Pro Val Lys Met Ile Arg Thr Met Met Ala

115 120 125 115 120 125

Gly Leu Ser Lys Tyr Glu Leu Asn Val Glu Ser Asn Ile Glu Leu IleGly Leu Ser Lys Tyr Glu Leu Asn Val Glu Ser Asn Ile Glu Leu Ile

130 135 140 130 135 140

Ile Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala SerIle Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser

145 150 155 160145 150 155 160

Ala Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg LysAla Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg Lys

165 170 175 165 170 175

Arg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ser Ile His Pro Glu TyrArg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ser Ile His Pro Glu Tyr

180 185 190 180 185 190

Lys Lys Thr Pro His Trp Phe Asp Arg Leu Asp Asn Gly Ser Ile GluLys Lys Thr Pro His Trp Phe Asp Arg Leu Asp Asn Gly Ser Ile Glu

195 200 205 195 200 205

Gly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly ValGly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly Val

210 215 220 210 215 220

Ser Glu Arg Thr Asn Lys Glu Ala Ile Ile Thr Ile Ala Lys His IleSer Glu Arg Thr Asn Lys Glu Ala Ile Ile Thr Ile Ala Lys His Ile

225 230 235 240225 230 235 240

Gln Asp Asn Lys Glu Ala Gln Phe Lys Lys Ile Val Ala Ile Asn ValGln Asp Asn Lys Glu Ala Gln Phe Lys Lys Ile Val Ala Ile Asn Val

245 250 255 245 250 255

Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met ValPro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Val

260 265 270 260 265 270

Asp Lys Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu LysAsp Lys Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu Lys

275 280 285 275 280 285

Val Trp Glu Ile Asp Leu Ser Lys Pro Ile Glu Met Val Glu Thr AsnVal Trp Glu Ile Asp Leu Ser Lys Pro Ile Glu Met Val Glu Thr Asn

290 295 300 290 295 300

Lys Pro Leu Ala Glu Val Leu Glu Ser Ile Ile Gly Glu Lys Pro IleLys Pro Leu Ala Glu Val Leu Glu Ser Ile Ile Gly Glu Lys Pro Ile

305 310 315 320305 310 315 320

Leu Ile Pro Ile Ala Gly Lys Asp Ala Thr Gln Leu Asp Ile Asp IleLeu Ile Pro Ile Ala Gly Lys Asp Ala Thr Gln Leu Asp Ile Asp Ile

325 330 335 325 330 335

Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly ValGlu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly Val

340 345 350 340 345 350

Val Val Gly Tyr Ser Arg Asn Val Lys Thr Glu Ala Ala Leu Arg AlaVal Val Gly Tyr Ser Arg Asn Val Lys Thr Glu Ala Ala Leu Arg Ala

355 360 365 355 360 365

Ala Gly Val Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu GlyAla Gly Val Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu Gly

370 375 380 370 375 380

Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp ValMet Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp Val

385 390 395 400385 390 395 400

LysLys

<210> 65<210> 65

<211> 401<211> 401

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 惰性支原体-火鸡支原体嵌合ADI<223> Laboratory Preparation - M. inert-M. turkeyi chimeric ADI

<400> 65<400> 65

Met Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Val Leu Lys Glu ValMet Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Val Leu Lys Glu Val

1 5 10 151 5 10 15

Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ala Pro Ser ArgLeu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ala Pro Ser Arg

20 25 30 20 25 30

Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Ser Ala Ala IleLeu Asp Glu Leu Leu Phe Ser Ala Ile Leu Glu Pro Ser Ala Ala Ile

35 40 45 35 40 45

Gln Glu His Lys Ser Phe Leu Lys Ile Leu Gln Asp Arg Gly Ile LysGln Glu His Lys Ser Phe Leu Lys Ile Leu Gln Asp Arg Gly Ile Lys

50 55 60 50 55 60

Thr Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Val Lys Tyr AlaThr Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Val Lys Tyr Ala

65 70 75 8065 70 75 80

Thr Ser Lys Glu Lys Glu Ser Phe Ile Glu Lys Trp Leu Asp Glu AlaThr Ser Lys Glu Lys Glu Ser Phe Ile Glu Lys Trp Leu Asp Glu Ala

85 90 95 85 90 95

Thr Pro Ala Leu Asn Ser Glu Asn Arg Ala Arg Val Lys Asn Tyr IleThr Pro Ala Leu Asn Ser Glu Asn Arg Ala Arg Val Lys Asn Tyr Ile

100 105 110 100 105 110

Thr Ala Met Gln Gly Gln Pro Val Lys Met Val Arg Ala Met Met AlaThr Ala Met Gln Gly Gln Pro Val Lys Met Val Arg Ala Met Met Ala

115 120 125 115 120 125

Gly Val Ser Lys Gln Glu Leu Asn Ile Glu Ser Asp Val Glu Leu IleGly Val Ser Lys Gln Glu Leu Asn Ile Glu Ser Asp Val Glu Leu Ile

130 135 140 130 135 140

Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala SerVal Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser

145 150 155 160145 150 155 160

Ala Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg LysAla Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg Lys

165 170 175 165 170 175

Arg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ser Ile His Pro Glu TyrArg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ser Ile His Pro Glu Tyr

180 185 190 180 185 190

Lys Lys Thr Pro His Trp Phe Asp Arg Leu Asp Asn Gly Ser Ile GluLys Lys Thr Pro His Trp Phe Asp Arg Leu Asp Asn Gly Ser Ile Glu

195 200 205 195 200 205

Gly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly ValGly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly Val

210 215 220 210 215 220

Ser Glu Arg Thr Asn Lys Glu Ala Ile Ile Thr Ile Ala Lys His IleSer Glu Arg Thr Asn Lys Glu Ala Ile Ile Thr Ile Ala Lys His Ile

225 230 235 240225 230 235 240

Gln Asp Asn Lys Glu Ala Gln Phe Lys Lys Ile Val Ala Ile Asn ValGln Asp Asn Lys Glu Ala Gln Phe Lys Lys Ile Val Ala Ile Asn Val

245 250 255 245 250 255

Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met ValPro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Val

260 265 270 260 265 270

Asp Lys Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu LysAsp Lys Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu Lys

275 280 285 275 280 285

Val Trp Glu Ile Asp Leu Ser Lys Pro Ile Glu Met Val Glu Thr AsnVal Trp Glu Ile Asp Leu Ser Lys Pro Ile Glu Met Val Glu Thr Asn

290 295 300 290 295 300

Lys Pro Leu Ala Glu Val Leu Glu Ser Ile Ile Gly Glu Lys Pro IleLys Pro Leu Ala Glu Val Leu Glu Ser Ile Ile Gly Glu Lys Pro Ile

305 310 315 320305 310 315 320

Leu Ile Pro Ile Ala Gly Lys Asp Ala Thr Gln Leu Asp Ile Asp IleLeu Ile Pro Ile Ala Gly Lys Asp Ala Thr Gln Leu Asp Ile Asp Ile

325 330 335 325 330 335

Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly ValGlu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly Val

340 345 350 340 345 350

Val Val Gly Tyr Ser Arg Asn Val Lys Thr Glu Ala Ala Leu Arg AlaVal Val Gly Tyr Ser Arg Asn Val Lys Thr Glu Ala Ala Leu Arg Ala

355 360 365 355 360 365

Ala Gly Val Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu GlyAla Gly Val Thr Val Leu Ser Phe Glu Gly Asn Gln Leu Ser Leu Gly

370 375 380 370 375 380

Met Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp ValMet Gly Ser Ala Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp Val

385 390 395 400385 390 395 400

LysLys

<210> 66<210> 66

<211> 401<211> 401

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 火鸡支原体-鸽支原体嵌合ADI<223> Lab Preparation - M. turkey-M. pigeon chimeric ADI

<400> 66<400> 66

Met Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Val Leu Lys Glu ValMet Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Val Leu Lys Glu Val

1 5 10 151 5 10 15

Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser ArgLeu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser Arg

20 25 30 20 25 30

Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Gln Pro Glu Gln Ala IleLeu Asp Glu Leu Leu Phe Ser Ala Ile Leu Gln Pro Glu Gln Ala Ile

35 40 45 35 40 45

Lys Glu His Gln Ser Phe Val Lys Ile Leu Gln Asp Arg Gly Ile LysLys Glu His Gln Ser Phe Val Lys Ile Leu Gln Asp Arg Gly Ile Lys

50 55 60 50 55 60

Val Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Thr Tyr His AlaVal Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Thr Tyr His Ala

65 70 75 8065 70 75 80

Thr Gln Lys Glu Arg Glu Ala Phe Ile Glu Lys Trp Leu Asp Glu AlaThr Gln Lys Glu Arg Glu Ala Phe Ile Glu Lys Trp Leu Asp Glu Ala

85 90 95 85 90 95

Glu Pro Ala Leu Thr Lys Asp Leu Arg Ala Lys Val Lys Ser Tyr ValGlu Pro Ala Leu Thr Lys Asp Leu Arg Ala Lys Val Lys Ser Tyr Val

100 105 110 100 105 110

Leu Ser Lys Glu Gly Thr Pro Val Ala Met Val Arg Thr Met Met AlaLeu Ser Lys Glu Gly Thr Pro Val Ala Met Val Arg Thr Met Met Ala

115 120 125 115 120 125

Gly Val Ser Lys Gln Glu Leu Asn Val Glu Ser Glu Thr Glu Leu ValGly Val Ser Lys Gln Glu Leu Asn Val Glu Ser Glu Thr Glu Leu Val

130 135 140 130 135 140

Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala SerVal Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser

145 150 155 160145 150 155 160

Ala Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg LysAla Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg Lys

165 170 175 165 170 175

Arg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ser Ile His Pro Glu TyrArg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ser Ile His Pro Glu Tyr

180 185 190 180 185 190

Lys Gln Thr Pro His Trp Phe Asp Arg Leu Asp Lys Gly Asn Ile GluLys Gln Thr Pro His Trp Phe Asp Arg Leu Asp Lys Gly Asn Ile Glu

195 200 205 195 200 205

Gly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly ValGly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly Val

210 215 220 210 215 220

Ser Glu Arg Thr Asn Lys Glu Ala Ile Leu Thr Ile Ala Glu His IleSer Glu Arg Thr Asn Lys Glu Ala Ile Leu Thr Ile Ala Glu His Ile

225 230 235 240225 230 235 240

Lys Asn Asn Lys Glu Ala Lys Phe Lys Lys Ile Val Ala Ile Asn ValLys Asn Asn Lys Glu Ala Lys Phe Lys Lys Ile Val Ala Ile Asn Val

245 250 255 245 250 255

Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met ValPro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Val

260 265 270 260 265 270

Asp Lys Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu LysAsp Lys Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu Lys

275 280 285 275 280 285

Ile Trp Glu Ile Asp Leu Ser Lys Glu Ile Lys Met Val Glu Thr SerIle Trp Glu Ile Asp Leu Ser Lys Glu Ile Lys Met Val Glu Thr Ser

290 295 300 290 295 300

Lys Pro Leu Ala Asp Val Leu Glu Ser Ile Ile Gly Glu Lys Pro IleLys Pro Leu Ala Asp Val Leu Glu Ser Ile Ile Gly Glu Lys Pro Ile

305 310 315 320305 310 315 320

Leu Ile Pro Ile Ala Gly Glu Asn Ala Ser Gln Leu Asp Ile Asp IleLeu Ile Pro Ile Ala Gly Glu Asn Ala Ser Gln Leu Asp Ile Asp Ile

325 330 335 325 330 335

Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly ValGlu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly Val

340 345 350 340 345 350

Val Val Gly Tyr Ser Arg Asn Val Lys Thr Glu Ala Ala Leu Lys AlaVal Val Gly Tyr Ser Arg Asn Val Lys Thr Glu Ala Ala Leu Lys Ala

355 360 365 355 360 365

Ala Gly Val Thr Val Tyr Ser Phe Asp Gly Asn Gln Leu Ser Leu GlyAla Gly Val Thr Val Tyr Ser Phe Asp Gly Asn Gln Leu Ser Leu Gly

370 375 380 370 375 380

Met Gly Ser Gly Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp ValMet Gly Ser Gly Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp Val

385 390 395 400385 390 395 400

LysLys

<210> 67<210> 67

<211> 401<211> 401

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 火鸡支原体-鸡支原体嵌合ADI<223> Laboratory Preparation - M. turkey-M. gallis chimeric ADI

<400> 67<400> 67

Met Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Val Leu Lys Glu ValMet Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Val Leu Lys Glu Val

1 5 10 151 5 10 15

Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser ArgLeu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser Arg

20 25 30 20 25 30

Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Gln Pro Glu Gln Ala IleLeu Asp Glu Leu Leu Phe Ser Ala Ile Leu Gln Pro Glu Gln Ala Ile

35 40 45 35 40 45

Lys Glu His Gln Ser Phe Val Lys Ile Leu Gln Asp Arg Gly Ile LysLys Glu His Gln Ser Phe Val Lys Ile Leu Gln Asp Arg Gly Ile Lys

50 55 60 50 55 60

Val Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Val Lys Tyr AlaVal Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Val Lys Tyr Ala

65 70 75 8065 70 75 80

Thr Ala Glu Gln Lys Ala Ala Phe Ile Glu Lys Tyr Leu Asp Glu AlaThr Ala Glu Gln Lys Ala Ala Phe Ile Glu Lys Tyr Leu Asp Glu Ala

85 90 95 85 90 95

Thr Pro Ala Leu Ser Ala Glu Asn Arg Glu Arg Ala Lys Lys Tyr IleThr Pro Ala Leu Ser Ala Glu Asn Arg Glu Arg Ala Lys Lys Tyr Ile

100 105 110 100 105 110

Leu Ser Leu Glu Met Gln Pro Val Lys Met Ile Arg Thr Met Met AlaLeu Ser Leu Glu Met Gln Pro Val Lys Met Ile Arg Thr Met Met Ala

115 120 125 115 120 125

Gly Leu Ser Lys Tyr Glu Leu Asn Val Glu Ser Asn Ile Glu Leu IleGly Leu Ser Lys Tyr Glu Leu Asn Val Glu Ser Asn Ile Glu Leu Ile

130 135 140 130 135 140

Ile Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala SerIle Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser

145 150 155 160145 150 155 160

Ala Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg LysAla Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg Lys

165 170 175 165 170 175

Arg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ser Ile His Pro Glu TyrArg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ser Ile His Pro Glu Tyr

180 185 190 180 185 190

Lys Gln Thr Pro His Trp Phe Asp Arg Leu Asp Lys Gly Asn Ile GluLys Gln Thr Pro His Trp Phe Asp Arg Leu Asp Lys Gly Asn Ile Glu

195 200 205 195 200 205

Gly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly ValGly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly Val

210 215 220 210 215 220

Ser Glu Arg Thr Asn Lys Glu Ala Ile Leu Thr Ile Ala Glu His IleSer Glu Arg Thr Asn Lys Glu Ala Ile Leu Thr Ile Ala Glu His Ile

225 230 235 240225 230 235 240

Lys Asn Asn Lys Glu Ala Lys Phe Lys Lys Ile Val Ala Ile Asn ValLys Asn Asn Lys Glu Ala Lys Phe Lys Lys Ile Val Ala Ile Asn Val

245 250 255 245 250 255

Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met ValPro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Val

260 265 270 260 265 270

Asp Lys Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu LysAsp Lys Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu Lys

275 280 285 275 280 285

Ile Trp Glu Ile Asp Leu Ser Lys Glu Ile Lys Met Val Glu Thr SerIle Trp Glu Ile Asp Leu Ser Lys Glu Ile Lys Met Val Glu Thr Ser

290 295 300 290 295 300

Lys Pro Leu Ala Asp Val Leu Glu Ser Ile Ile Gly Glu Lys Pro IleLys Pro Leu Ala Asp Val Leu Glu Ser Ile Ile Gly Glu Lys Pro Ile

305 310 315 320305 310 315 320

Leu Ile Pro Ile Ala Gly Glu Asn Ala Ser Gln Leu Asp Ile Asp IleLeu Ile Pro Ile Ala Gly Glu Asn Ala Ser Gln Leu Asp Ile Asp Ile

325 330 335 325 330 335

Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly ValGlu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly Val

340 345 350 340 345 350

Val Val Gly Tyr Ser Arg Asn Val Lys Thr Glu Ala Ala Leu Lys AlaVal Val Gly Tyr Ser Arg Asn Val Lys Thr Glu Ala Ala Leu Lys Ala

355 360 365 355 360 365

Ala Gly Val Thr Val Tyr Ser Phe Asp Gly Asn Gln Leu Ser Leu GlyAla Gly Val Thr Val Tyr Ser Phe Asp Gly Asn Gln Leu Ser Leu Gly

370 375 380 370 375 380

Met Gly Ser Gly Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp ValMet Gly Ser Gly Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp Val

385 390 395 400385 390 395 400

LysLys

<210> 68<210> 68

<211> 401<211> 401

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 火鸡支原体-惰性支原体嵌合ADI<223> Laboratory Preparation - M. turkeyi-M. inert chimeric ADI

<400> 68<400> 68

Met Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Val Leu Lys Glu ValMet Ser Lys Ile Asn Val Tyr Ser Glu Ile Gly Val Leu Lys Glu Val

1 5 10 151 5 10 15

Leu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser ArgLeu Val His Thr Pro Gly Asp Glu Ile Arg Arg Ile Ser Pro Ser Arg

20 25 30 20 25 30

Leu Asp Glu Leu Leu Phe Ser Ala Ile Leu Gln Pro Glu Gln Ala IleLeu Asp Glu Leu Leu Phe Ser Ala Ile Leu Gln Pro Glu Gln Ala Ile

35 40 45 35 40 45

Lys Glu His Gln Ser Phe Val Lys Ile Leu Gln Asp Arg Gly Ile LysLys Glu His Gln Ser Phe Val Lys Ile Leu Gln Asp Arg Gly Ile Lys

50 55 60 50 55 60

Val Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Lys His Tyr AlaVal Ile Gln Leu Ser Asp Leu Val Ala Glu Thr Tyr Lys His Tyr Ala

65 70 75 8065 70 75 80

Ser Glu Ala Glu Lys Glu Ala Phe Ile Glu Lys Tyr Leu Asp Glu AlaSer Glu Ala Glu Lys Glu Ala Phe Ile Glu Lys Tyr Leu Asp Glu Ala

85 90 95 85 90 95

Thr Pro Val Leu Ser Lys Asp Met Arg Ala Lys Val Lys Asn Tyr IleThr Pro Val Leu Ser Lys Asp Met Arg Ala Lys Val Lys Asn Tyr Ile

100 105 110 100 105 110

Leu Ser Met Gln Gly Glu Pro Val Lys Met Val Arg Thr Met Met AlaLeu Ser Met Gln Gly Glu Pro Val Lys Met Val Arg Thr Met Met Ala

115 120 125 115 120 125

Gly Val Ser Lys Gln Glu Leu Asn Val Glu Ser Glu Val Glu Leu IleGly Val Ser Lys Gln Glu Leu Asn Val Glu Ser Glu Val Glu Leu Ile

130 135 140 130 135 140

Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala SerVal Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser

145 150 155 160145 150 155 160

Ala Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg LysAla Gly Asn Gly Ile Ser Leu Asn Asn Met Lys Tyr Val Val Arg Lys

165 170 175 165 170 175

Arg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ser Ile His Pro Glu TyrArg Glu Thr Ile Phe Ala Glu Phe Ile Phe Ser Ile His Pro Glu Tyr

180 185 190 180 185 190

Lys Gln Thr Pro His Trp Phe Asp Arg Leu Asp Lys Gly Asn Ile GluLys Gln Thr Pro His Trp Phe Asp Arg Leu Asp Lys Gly Asn Ile Glu

195 200 205 195 200 205

Gly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly ValGly Gly Asp Val Phe Ile Tyr Asn Lys Asp Thr Leu Val Ile Gly Val

210 215 220 210 215 220

Ser Glu Arg Thr Asn Lys Glu Ala Ile Leu Thr Ile Ala Glu His IleSer Glu Arg Thr Asn Lys Glu Ala Ile Leu Thr Ile Ala Glu His Ile

225 230 235 240225 230 235 240

Lys Asn Asn Lys Glu Ala Lys Phe Lys Lys Ile Val Ala Ile Asn ValLys Asn Asn Lys Glu Ala Lys Phe Lys Lys Ile Val Ala Ile Asn Val

245 250 255 245 250 255

Pro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met ValPro Pro Met Pro Asn Leu Met His Leu Asp Thr Trp Leu Thr Met Val

260 265 270 260 265 270

Asp Lys Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu LysAsp Lys Asn Lys Phe Leu Tyr Ser Pro Asn Met Leu Ser Val Leu Lys

275 280 285 275 280 285

Ile Trp Glu Ile Asp Leu Ser Lys Glu Ile Lys Met Val Glu Thr SerIle Trp Glu Ile Asp Leu Ser Lys Glu Ile Lys Met Val Glu Thr Ser

290 295 300 290 295 300

Lys Pro Leu Ala Asp Val Leu Glu Ser Ile Ile Gly Glu Lys Pro IleLys Pro Leu Ala Asp Val Leu Glu Ser Ile Ile Gly Glu Lys Pro Ile

305 310 315 320305 310 315 320

Leu Ile Pro Ile Ala Gly Glu Asn Ala Ser Gln Leu Asp Ile Asp IleLeu Ile Pro Ile Ala Gly Glu Asn Ala Ser Gln Leu Asp Ile Asp Ile

325 330 335 325 330 335

Glu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly ValGlu Thr His Phe Asp Gly Thr Asn Tyr Leu Thr Ile Ala Pro Gly Val

340 345 350 340 345 350

Val Val Gly Tyr Ser Arg Asn Val Lys Thr Glu Ala Ala Leu Lys AlaVal Val Gly Tyr Ser Arg Asn Val Lys Thr Glu Ala Ala Leu Lys Ala

355 360 365 355 360 365

Ala Gly Val Thr Val Tyr Ser Phe Asp Gly Asn Gln Leu Ser Leu GlyAla Gly Val Thr Val Tyr Ser Phe Asp Gly Asn Gln Leu Ser Leu Gly

370 375 380 370 375 380

Met Gly Ser Gly Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp ValMet Gly Ser Gly Arg Cys Met Ser Met Pro Leu Val Arg Glu Asp Val

385 390 395 400385 390 395 400

LysLys

<210> 69<210> 69

<211> 281<211> 281

<212> PRT<212> PRT

<213> 智人（Homo sapiens）<213> Homo sapiens

<400> 69<400> 69

Met Ala Met Met Glu Val Gln Gly Gly Pro Ser Leu Gly Gln Thr CysMet Ala Met Met Glu Val Gln Gly Gly Pro Ser Leu Gly Gln Thr Cys

1 5 10 151 5 10 15

Val Leu Ile Val Ile Phe Thr Val Leu Leu Gln Ser Leu Cys Val AlaVal Leu Ile Val Ile Phe Thr Val Leu Leu Gln Ser Leu Cys Val Ala

20 25 30 20 25 30

Val Thr Tyr Val Tyr Phe Thr Asn Glu Leu Lys Gln Met Gln Asp LysVal Thr Tyr Val Tyr Phe Thr Asn Glu Leu Lys Gln Met Gln Asp Lys

35 40 45 35 40 45

Tyr Ser Lys Ser Gly Ile Ala Cys Phe Leu Lys Glu Asp Asp Ser TyrTyr Ser Lys Ser Gly Ile Ala Cys Phe Leu Lys Glu Asp Asp Ser Tyr

50 55 60 50 55 60

Trp Asp Pro Asn Asp Glu Glu Ser Met Asn Ser Pro Cys Trp Gln ValTrp Asp Pro Asn Asp Glu Glu Ser Met Asn Ser Pro Cys Trp Gln Val

65 70 75 8065 70 75 80

Lys Trp Gln Leu Arg Gln Leu Val Arg Lys Met Ile Leu Arg Thr SerLys Trp Gln Leu Arg Gln Leu Val Arg Lys Met Ile Leu Arg Thr Ser

85 90 95 85 90 95

Glu Glu Thr Ile Ser Thr Val Gln Glu Lys Gln Gln Asn Ile Ser ProGlu Glu Thr Ile Ser Thr Val Gln Glu Lys Gln Gln Asn Ile Ser Pro

100 105 110 100 105 110

Leu Val Arg Glu Arg Gly Pro Gln Arg Val Ala Ala His Ile Thr GlyLeu Val Arg Glu Arg Gly Pro Gln Arg Val Ala Ala His Ile Thr Gly

115 120 125 115 120 125

Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn GluThr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu

130 135 140 130 135 140

Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg Ser GlyLys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly

145 150 155 160145 150 155 160

His Ser Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu Leu Val IleHis Ser Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile

165 170 175 165 170 175

His Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg PheHis Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe

180 185 190 180 185 190

Gln Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val GlnGln Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln

195 200 205 195 200 205

Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met LysTyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys

210 215 220 210 215 220

Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu TyrSer Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr

225 230 235 240225 230 235 240

Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg IleSer Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile

245 250 255 245 250 255

Phe Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His Glu AlaPhe Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His Glu Ala

260 265 270 260 265 270

Ser Phe Phe Gly Ala Phe Leu Val GlySer Phe Phe Gly Ala Phe Leu Val Gly

275 280 275 280

<210> 70<210> 70

<211> 168<211> 168

<212> PRT<212> PRT

<213> 智人（Homo sapiens）<213> Homo sapiens

<400> 70<400> 70

Val Arg Glu Arg Gly Pro Gln Arg Val Ala Ala His Ile Thr Gly ThrVal Arg Glu Arg Gly Pro Gln Arg Val Ala Ala His Ile Thr Gly Thr

1 5 10 151 5 10 15

Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu LysArg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu Lys

20 25 30 20 25 30

Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly HisAla Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly His

35 40 45 35 40 45

Ser Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile HisSer Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile His

50 55 60 50 55 60

Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe GlnGlu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe Gln

65 70 75 8065 70 75 80

Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln TyrGlu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln Tyr

85 90 95 85 90 95

Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys SerIle Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys Ser

100 105 110 100 105 110

Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr SerAla Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr Ser

115 120 125 115 120 125

Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile PheIle Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile Phe

130 135 140 130 135 140

Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His Glu Ala SerVal Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His Glu Ala Ser

145 150 155 160145 150 155 160

Phe Phe Gly Ala Phe Leu Val GlyPhe Phe Gly Ala Phe Leu Val Gly

165 165

<210> 71<210> 71

<211> 233<211> 233

<212> PRT<212> PRT

<213> 智人（Homo sapiens）<213> Homo sapiens

<400> 71<400> 71

Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu AlaMet Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala

1 5 10 151 5 10 15

Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu PheLeu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe

20 25 30 20 25 30

Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr Leu PheLeu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr Leu Phe

35 40 45 35 40 45

Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu Glu Phe ProCys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu Glu Phe Pro

50 55 60 50 55 60

Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln Ala Val Arg Ser SerArg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln Ala Val Arg Ser Ser

65 70 75 8065 70 75 80

Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn ProSer Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro

85 90 95 85 90 95

Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala LeuGln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu

100 105 110 100 105 110

Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro SerLeu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser

115 120 125 115 120 125

Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln GlyGlu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly

130 135 140 130 135 140

Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile AlaCys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala

145 150 155 160145 150 155 160

Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser ProVal Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro

165 170 175 165 170 175

Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr GluCys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu

180 185 190 180 185 190

Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg LeuPro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu

195 200 205 195 200 205

Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser GlySer Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly

210 215 220 210 215 220

Gln Val Tyr Phe Gly Ile Ile Ala LeuGln Val Tyr Phe Gly Ile Ile Ala Leu

225 230225 230

<210> 72<210> 72

<211> 281<211> 281

<212> PRT<212> PRT

<213> 智人（Homo sapiens）<213> Homo sapiens

<400> 72<400> 72

Met Gln Gln Pro Phe Asn Tyr Pro Tyr Pro Gln Ile Tyr Trp Val AspMet Gln Gln Pro Phe Asn Tyr Pro Tyr Pro Gln Ile Tyr Trp Val Asp

1 5 10 151 5 10 15

Ser Ser Ala Ser Ser Pro Trp Ala Pro Pro Gly Thr Val Leu Pro CysSer Ser Ala Ser Ser Pro Trp Ala Pro Pro Gly Thr Val Leu Pro Cys

20 25 30 20 25 30

Pro Thr Ser Val Pro Arg Arg Pro Gly Gln Arg Arg Pro Pro Pro ProPro Thr Ser Val Pro Arg Arg Pro Gly Gln Arg Arg Pro Pro Pro Pro

35 40 45 35 40 45

Pro Pro Pro Pro Pro Leu Pro Pro Pro Pro Pro Pro Pro Pro Leu ProPro Pro Pro Pro Pro Leu Pro Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro

50 55 60 50 55 60

Pro Leu Pro Leu Pro Pro Leu Lys Lys Arg Gly Asn His Ser Thr GlyPro Leu Pro Leu Pro Pro Leu Lys Lys Arg Gly Asn His Ser Thr Gly

65 70 75 8065 70 75 80

Leu Cys Leu Leu Val Met Phe Phe Met Val Leu Val Ala Leu Val GlyLeu Cys Leu Leu Val Met Phe Phe Met Val Leu Val Ala Leu Val Gly

85 90 95 85 90 95

Leu Gly Leu Gly Met Phe Gln Leu Phe His Leu Gln Lys Glu Leu AlaLeu Gly Leu Gly Met Phe Gln Leu Phe His Leu Gln Lys Glu Leu Ala

100 105 110 100 105 110

Glu Leu Arg Glu Ser Thr Ser Gln Met His Thr Ala Ser Ser Leu GluGlu Leu Arg Glu Ser Thr Ser Gln Met His Thr Ala Ser Ser Leu Glu

115 120 125 115 120 125

Lys Gln Ile Gly His Pro Ser Pro Pro Pro Glu Lys Lys Glu Leu ArgLys Gln Ile Gly His Pro Ser Pro Pro Pro Glu Lys Lys Glu Leu Arg

130 135 140 130 135 140

Lys Val Ala His Leu Thr Gly Lys Ser Asn Ser Arg Ser Met Pro LeuLys Val Ala His Leu Thr Gly Lys Ser Asn Ser Arg Ser Met Pro Leu

145 150 155 160145 150 155 160

Glu Trp Glu Asp Thr Tyr Gly Ile Val Leu Leu Ser Gly Val Lys TyrGlu Trp Glu Asp Thr Tyr Gly Ile Val Leu Leu Ser Gly Val Lys Tyr

165 170 175 165 170 175

Lys Lys Gly Gly Leu Val Ile Asn Glu Thr Gly Leu Tyr Phe Val TyrLys Lys Gly Gly Leu Val Ile Asn Glu Thr Gly Leu Tyr Phe Val Tyr

180 185 190 180 185 190

Ser Lys Val Tyr Phe Arg Gly Gln Ser Cys Asn Asn Leu Pro Leu SerSer Lys Val Tyr Phe Arg Gly Gln Ser Cys Asn Asn Leu Pro Leu Ser

195 200 205 195 200 205

His Lys Val Tyr Met Arg Asn Ser Lys Tyr Pro Gln Asp Leu Val MetHis Lys Val Tyr Met Arg Asn Ser Lys Tyr Pro Gln Asp Leu Val Met

210 215 220 210 215 220

Met Glu Gly Lys Met Met Ser Tyr Cys Thr Thr Gly Gln Met Trp AlaMet Glu Gly Lys Met Met Met Ser Tyr Cys Thr Thr Gly Gln Met Trp Ala

225 230 235 240225 230 235 240

Arg Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr Ser Ala Asp HisArg Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr Ser Ala Asp His

245 250 255 245 250 255

Leu Tyr Val Asn Val Ser Glu Leu Ser Leu Val Asn Phe Glu Glu SerLeu Tyr Val Asn Val Ser Glu Leu Ser Leu Val Asn Phe Glu Glu Ser

260 265 270 260 265 270

Gln Thr Phe Phe Gly Leu Tyr Lys LeuGln Thr Phe Phe Gly Leu Tyr Lys Leu

275 280 275 280

<210> 73<210> 73

<211> 4<211> 4

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 连接子<223> Laboratory Preparation - Linkers

<400> 73<400> 73

Gly Ser Gly SerGly Ser Gly Ser

11

<210> 74<210> 74

<211> 4<211> 4

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 连接子<223> Laboratory Preparation - Linkers

<400> 74<400> 74

Gly Gly Ser GlyGly Gly Ser Gly

11

<210> 75<210> 75

<211> 4<211> 4

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 连接子<223> Laboratory Preparation - Linkers

<400> 75<400> 75

Gly Gly Gly SerGly Gly Gly Ser

11

<210> 76<210> 76

<211> 5<211> 5

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 连接子<223> Laboratory Preparation - Linkers

<400> 76<400> 76

Gly Gly Gly Gly SerGly Gly Gly Gly Ser

1 51 5

<210> 77<210> 77

<211> 4<211> 4

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 连接子<223> Laboratory Preparation - Linkers

<400> 77<400> 77

Gly Asn Gly AsnGly Asn Gly Asn

11

<210> 78<210> 78

<211> 4<211> 4

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 连接子<223> Laboratory Preparation - Linkers

<400> 78<400> 78

Gly Gly Asn GlyGly Gly Asn Gly

11

<210> 79<210> 79

<211> 4<211> 4

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 连接子<223> Laboratory Preparation - Linkers

<400> 79<400> 79

Gly Gly Gly AsnGly Gly Gly Asn

11

<210> 80<210> 80

<211> 5<211> 5

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 连接子<223> Laboratory Preparation - Linkers

<400> 80<400> 80

Gly Gly Gly Gly AsnGly Gly Gly Gly Asn

1 51 5

<210> 81<210> 81

<211> 7<211> 7

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 连接子<223> Laboratory Preparation - Linkers

<400> 81<400> 81

Ala Glu Ala Ala Ala Lys AlaAla Glu Ala Ala Ala Lys Ala

1 51 5

<210> 82<210> 82

<211> 12<211> 12

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 连接子<223> Laboratory Preparation - Linkers

<400> 82<400> 82

Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys AlaAla Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala

1 5 101 5 10

<210> 83<210> 83

<211> 6<211> 6

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 连接子<223> Laboratory Preparation - Linkers

<400> 83<400> 83

Ala Pro Ala Pro Lys ProAla Pro Ala Pro Lys Pro

1 51 5

<210> 84<210> 84

<211> 12<211> 12

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 连接子<223> Laboratory Preparation - Linkers

<400> 84<400> 84

Ala Pro Ala Pro Lys Pro Glu Pro Ala Pro Lys ProAla Pro Ala Pro Lys Pro Glu Pro Ala Pro Lys Pro

1 5 101 5 10

<210> 85<210> 85

<211> 10<211> 10

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 连接子<223> Laboratory Preparation - Linkers

<400> 85<400> 85

Gly Gly Gly Gly Ser Gly Gly Gly Gly SerGly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 101 5 10

<210> 86<210> 86

<211> 5<211> 5

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 连接子<223> Laboratory Preparation - Linkers

<400> 86<400> 86

Asp Gly Gly Gly SerAsp Gly Gly Gly Ser

1 51 5

<210> 87<210> 87

<211> 5<211> 5

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 连接子<223> Laboratory Preparation - Linkers

<400> 87<400> 87

Thr Gly Glu Lys ProThr Gly Glu Lys Pro

1 51 5

<210> 88<210> 88

<211> 4<211> 4

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 连接子<223> Laboratory Preparation - Linkers

<400> 88<400> 88

Gly Gly Arg ArgGly Gly Arg Arg

11

<210> 89<210> 89

<211> 14<211> 14

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 连接子<223> Laboratory Preparation - Linkers

<400> 89<400> 89

Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Val AspGlu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Val Asp

1 5 101 5 10

<210> 90<210> 90

<211> 18<211> 18

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 连接子<223> Laboratory Preparation - Linkers

<400> 90<400> 90

Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg SerLys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser

1 5 10 151 5 10 15

Leu AspLeu Asp

<210> 91<210> 91

<211> 8<211> 8

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 连接子<223> Laboratory Preparation - Linkers

<400> 91<400> 91

Gly Gly Arg Arg Gly Gly Gly SerGly Gly Arg Arg Gly Gly Gly Ser

1 51 5

<210> 92<210> 92

<211> 9<211> 9

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 连接子<223> Laboratory Preparation - Linkers

<400> 92<400> 92

Leu Arg Gln Arg Asp Gly Glu Arg ProLeu Arg Gln Arg Asp Gly Glu Arg Pro

1 51 5

<210> 93<210> 93

<211> 12<211> 12

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 连接子<223> Laboratory Preparation - Linkers

<400> 93<400> 93

Leu Arg Gln Lys Asp Gly Gly Gly Ser Glu Arg ProLeu Arg Gln Lys Asp Gly Gly Gly Ser Glu Arg Pro

1 5 101 5 10

<210> 94<210> 94

<211> 16<211> 16

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 连接子<223> Laboratory Preparation - Linkers

<400> 94<400> 94

Leu Arg Gln Lys Asp Gly Gly Gly Ser Gly Gly Gly Ser Glu Arg ProLeu Arg Gln Lys Asp Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Arg Pro

1 5 10 151 5 10 15

<210> 95<210> 95

<211> 10<211> 10

<212> DNA<212> DNA

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 共有Kozak序列<223> consensus Kozak sequence

<400> 95<400> 95

rccrccatgg 10rccrccatgg 10

<210> 96<210> 96

<211> 6<211> 6

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 连接子<223> Laboratory Preparation - Linkers

<220><220>

<221> MOD_RES<221> MOD_RES

<222> (1)..(1)<222> (1)..(1)

<223> Xaa是任何氨基酸<223> Xaa is any amino acid

<220><220>

<221> MOD_RES<221> MOD_RES

<222> (3)..(3)<222> (3)..(3)

<223> Xaa是任何氨基酸<223> Xaa is any amino acid

<220><220>

<221> MOD_RES<221> MOD_RES

<222> (5)..(5)<222> (5)..(5)

<223> Xaa是任何氨基酸<223> Xaa is any amino acid

<400> 96<400> 96

Xaa Pro Xaa Pro Xaa ProXaa Pro Xaa Pro Xaa Pro

1 51 5

<210> 97<210> 97

<211> 12<211> 12

<212> PRT<212> PRT

<213> 人工序列（Artificial Sequence）<213> Artificial Sequence

<220><220>

<223> 实验室制备 - 连接子<223> Laboratory Preparation - Linkers

<220><220>

<221> MOD_RES<221> MOD_RES

<222> (1)..(1)<222> (1)..(1)

<223> Xaa是任何氨基酸<223> Xaa is any amino acid

<220><220>

<221> MOD_RES<221> MOD_RES

<222> (3)..(3)<222> (3)..(3)

<223> Xaa是任何氨基酸<223> Xaa is any amino acid

<220><220>

<221> MOD_RES<221> MOD_RES

<222> (5)..(5)<222> (5)..(5)

<223> Xaa是任何氨基酸<223> Xaa is any amino acid

<220><220>

<221> MOD_RES<221> MOD_RES

<222> (7)..(7)<222> (7)..(7)

<223> Xaa是任何氨基酸<223> Xaa is any amino acid

<220><220>

<221> MOD_RES<221> MOD_RES

<222> (9)..(9)<222> (9)..(9)

<223> Xaa是任何氨基酸<223> Xaa is any amino acid

<220><220>

<221> MOD_RES<221> MOD_RES

<222> (11)..(11)<222> (11)..(11)

<223> Xaa是任何氨基酸<223> Xaa is any amino acid

<400> 97<400> 97

Xaa Pro Xaa Pro Xaa Pro Xaa Pro Xaa Pro Xaa ProXaa Pro Xaa Pro Xaa Pro Xaa Pro Xaa Pro Xaa Pro

1 5 101 5 10

Claims (43)

Translated fromChinese

1.一种缀合物，其包括共价连接到肿瘤坏死因子(TNF)超家族配体的精氨酸脱亚胺酶(ADI)。WHAT IS CLAIMED IS: 1. A conjugate comprising an arginine deiminase (ADI) covalently linked to a tumor necrosis factor (TNF) superfamily ligand.2.根据权利要求1所述的缀合物，其中所述ADI包括与选自表A1的序列至少90％、95％、96％、97％、98％或99％相同的氨基酸序列，由所述氨基酸序列组成或基本上由所述氨基酸序列组成。2. The conjugate of claim 1, wherein the ADI comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to a sequence selected from Table A1, The amino acid sequence consists of or consists essentially of the amino acid sequence.3.根据权利要求1或2所述的缀合物，其中所述ADI是六聚体ADI多肽。3. The conjugate of claim 1 or 2, wherein the ADI is a hexameric ADI polypeptide.4.根据权利要求3所述的缀合物，其中所述六聚体ADI包括与SEQ ID NO:9、37、38、50或57-68至少90％、95％、96％、97％、98％或99％相同的氨基酸序列，由所述氨基酸序列组成或基本上由所述氨基酸序列组成。4. The conjugate of claim 3, wherein the hexameric ADI comprises at least 90%, 95%, 96%, 97%, SEQ ID NO: 9, 37, 38, 50 or 57-68 Amino acid sequences that are 98% or 99% identical, consist of, or consist essentially of, said amino acid sequences.5.根据前述权利要求中任一项所述的缀合物，其中所述TNF超家族配体选自表T1。5. The conjugate of any preceding claim, wherein the TNF superfamily ligand is selected from Table Tl.6.根据权利要求5所述的缀合物，其中所述超家族配体选自TNF相关的凋亡诱导配体(TRAIL)、TNF-α和FasL。6. The conjugate of claim 5, wherein the superfamily ligand is selected from the group consisting of TNF-related apoptosis-inducing ligand (TRAIL), TNF-alpha and FasL.7.根据权利要求6所述的缀合物，其中所述TNF超家族配体包括与选自表T2的序列至少90％、95％、96％、97％、98％或99％相同的氨基酸序列，由所述氨基酸序列组成或基本上由所述氨基酸序列组成。7. The conjugate of claim 6, wherein the TNF superfamily ligand comprises amino acids that are at least 90%, 95%, 96%, 97%, 98% or 99% identical to a sequence selected from Table T2 sequence, consisting of or consisting essentially of the amino acid sequence.8.根据前述权利要求中任一项所述的缀合物，其中所述TNF超家族配体是三聚体或同源三聚体多肽。8. The conjugate of any preceding claim, wherein the TNF superfamily ligand is a trimeric or homotrimeric polypeptide.9.根据前述权利要求中任一项所述的缀合物，其中所述ADI和所述TNF超家族配体通过连接子，任选地生理稳定的连接子分开。9. The conjugate of any preceding claim, wherein the ADI and the TNF superfamily ligand are separated by a linker, optionally a physiologically stable linker.10.根据权利要求9所述的缀合物，其中所述连接子为肽连接子，任选地为柔性肽连接子或刚性肽连接子。10. The conjugate of claim 9, wherein the linker is a peptide linker, optionally a flexible peptide linker or a rigid peptide linker.11.根据权利要求10所述的缀合物，其中所述肽连接子的长度为约1-100个氨基酸、约1-90个氨基酸、约1-80个氨基酸、约1-70个氨基酸、约1-80个氨基酸、约1-50个氨基酸、约1-40个氨基酸、约1-30个氨基酸、约1-20个氨基酸、约1-10个氨基酸或约1-5个氨基酸，或长度为约1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、60、70、80、90或100个氨基酸。11. The conjugate of claim 10, wherein the peptide linker is about 1-100 amino acids, about 1-90 amino acids, about 1-80 amino acids, about 1-70 amino acids in length, about 1-80 amino acids, about 1-50 amino acids, about 1-40 amino acids, about 1-30 amino acids, about 1-20 amino acids, about 1-10 amino acids, or about 1-5 amino acids, or Lengths are about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 60, 70, 80, 90 or 100 amino acids.12.根据权利要求9或10所述的缀合物，其中所述肽连接子选自表L1。12. The conjugate of claim 9 or 10, wherein the peptide linker is selected from Table L1.13.根据前述权利要求中任一项所述的缀合物，其中所述缀合物是融合多肽。13. The conjugate of any preceding claim, wherein the conjugate is a fusion polypeptide.14.根据权利要求13所述的缀合物，其中所述ADI融合到所述TNF超家族配体的N端，任选地通过连接子分开。14. The conjugate of claim 13, wherein the ADI is fused to the N-terminus of the TNF superfamily ligand, optionally separated by a linker.15.根据权利要求13所述的缀合物，其中所述ADI融合到所述TNF超家族配体的C端，任选地通过连接子分开。15. The conjugate of claim 13, wherein the ADI is fused to the C-terminus of the TNF superfamily ligand, optionally separated by a linker.16.根据权利要求9所述的缀合物，其中所述连接子为非肽连接子。16. The conjugate of claim 9, wherein the linker is a non-peptide linker.17.根据前述权利要求中任一项所述的缀合物，其中所述缀合物相对于单独的所述ADI和/或单独的所述TNF超家族配体具有改善的药代动力学、物理和/或生物学性质，任选地选自增加的稳定性、增加的血清半衰期、提高的生物利用度、增加的生物活性、增加的暴露度和降低的清除率中的一种或多种。17. The conjugate of any preceding claim, wherein the conjugate has improved pharmacokinetics, relative to the ADI alone and/or the TNF superfamily ligand alone, Physical and/or biological properties, optionally selected from one or more of increased stability, increased serum half-life, increased bioavailability, increased biological activity, increased exposure, and decreased clearance .18.根据权利要求17所述的缀合物，其中所述缀合物相对于单独的所述ADI和/或单独的所述TNF超家族配体具有增加的稳定性和/或血清半衰期，任选地其中与所述缀合物相关的所述稳定性和/或血清半衰期相对于单独的所述ADI和/或单独的所述TNF超家族配体增加了约或至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、200％、300％、400％、500％、600％、700％、800％、900％或1000％或更多。18. The conjugate of claim 17, wherein the conjugate has increased stability and/or serum half-life relative to the ADI alone and/or the TNF superfamily ligand alone, any Optionally wherein said stability and/or serum half-life associated with said conjugate is increased by about or at least about 10%, 20% relative to said ADI alone and/or said TNF superfamily ligand alone , 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000 %Or more.19.根据权利要求17所述的缀合物，其中所述缀合物相对于单独的所述ADI和/或单独的所述TNF超家族配体具有增加的生物活性，任选地其中所述缀合物的生物活性相对于单独的所述ADI和/或单独的所述TNF超家族配体增加了约或至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、200％、300％、400％、500％、600％、700％、800％、900％或1000％或更多，或任选地其中所述生物活性相对于单独的所述ADI和/或单独的所述TNF超家族配体协同增加。19. The conjugate of claim 17, wherein the conjugate has increased biological activity relative to the ADI alone and/or the TNF superfamily ligand alone, optionally wherein the The biological activity of the conjugate is increased by about or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70% relative to the ADI alone and/or the TNF superfamily ligand alone %, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% or more, or optionally wherein the biologically active Synergistically increased relative to the ADI alone and/or the TNF superfamily ligand alone.20.根据权利要求19所述的缀合物，其中所述生物活性是在癌细胞中诱导细胞死亡或凋亡，所述诱导相对于单独的所述ADI和/或单独的所述TNF超家族配体任选地协同增加。20. The conjugate of claim 19, wherein the biological activity is induction of cell death or apoptosis in cancer cells relative to the ADI alone and/or the TNF superfamily alone Ligands are optionally added synergistically.21.根据权利要求20所述的缀合物，其中所述癌细胞是ADI敏感的细胞，其任选地选自乳腺癌细胞、肝细胞癌细胞、伯基特淋巴瘤细胞、结肠癌细胞、胶质母细胞瘤癌细胞、白血病细胞、黑色素瘤癌细胞、非小细胞肺癌(NSCLC)细胞、卵巢癌细胞、胰腺癌细胞、前列腺癌细胞和肾癌细胞中的一种或多种。21. The conjugate of claim 20, wherein the cancer cells are ADI-sensitive cells optionally selected from breast cancer cells, hepatocellular carcinoma cells, Burkitt lymphoma cells, colon cancer cells, One or more of glioblastoma cancer cells, leukemia cells, melanoma cancer cells, non-small cell lung cancer (NSCLC) cells, ovarian cancer cells, pancreatic cancer cells, prostate cancer cells, and kidney cancer cells.22.根据权利要求20所述的缀合物，其中所述癌细胞是ADI不敏感的细胞，其任选地选自乳腺癌细胞、结肠癌细胞和NSCLC细胞中的一种或多种。22. The conjugate of claim 20, wherein the cancer cells are ADI-insensitive cells, optionally selected from one or more of breast cancer cells, colon cancer cells, and NSCLC cells.23.根据权利要求19-22中任一项所述的缀合物，其中相对于单独的所述TNF超家族配体，所述ADI任选地使所述TNF超家族配体诱导癌细胞中细胞死亡或凋亡的能力增加约至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、200％、300％、400％、500％、600％、700％、800％、900％或1000％。23. The conjugate of any one of claims 19-22, wherein the ADI optionally causes the TNF superfamily ligand to induce in cancer cells relative to the TNF superfamily ligand alone. Increased ability of cells to die or apoptosis by about at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000%.24.根据权利要求23的缀合物，其中所述ADI上调所述癌细胞上死亡受体5(DR5)的表达。24. The conjugate according to claim 23, wherein the ADI upregulates the expression of death receptor 5 (DR5) on the cancer cells.25.根据前述权利要求中任一项所述的缀合物，其中所述ADI多肽通过连接基团共价键合到至少一个聚乙二醇(PEG)分子，任选地其中所述TNF超家族配体不共价键合到PEG分子。25. The conjugate of any one of the preceding claims, wherein the ADI polypeptide is covalently bonded to at least one polyethylene glycol (PEG) molecule through a linking group, optionally wherein the TNF supernatant is Family ligands are not covalently bonded to PEG molecules.26.一种缀合物，其包括(a)共价连接到三聚体多肽的六聚体多肽，或(b)共价连接到第二三聚体多肽的第一三聚体多肽，所述第二三聚体多肽与所述第一三聚体多肽不同。26. A conjugate comprising (a) a hexameric polypeptide covalently linked to a trimeric polypeptide, or (b) a first trimeric polypeptide covalently linked to a second trimeric polypeptide, wherein The second trimeric polypeptide is different from the first trimeric polypeptide.27.根据权利要求26所述的缀合物，其中所述六聚体多肽是同源六聚体多肽。27. The conjugate of claim 26, wherein the hexameric polypeptide is a homohexameric polypeptide.28.根据权利要求26或27所述的缀合物，其中所述六聚体多肽选自精氨酸脱亚胺酶，任选地如前述权利要求中任一项或多项所定义的精氨酸脱亚胺酶，以及脂联素或其胶原样结构域。28. The conjugate according to claim 26 or 27, wherein the hexameric polypeptide is selected from arginine deiminase, optionally an arginine as defined in any one or more of the preceding claims. amino acid deiminase, and adiponectin or its collagen-like domain.29.根据权利要求26-28中任一项所述的缀合物，其中所述三聚体多肽是同源三聚体多肽。29. The conjugate of any one of claims 26-28, wherein the trimeric polypeptide is a homotrimeric polypeptide.30.根据权利要求26-29中任一项所述的缀合物，其中(b)的所述第一三聚体多肽选自脂联素或其胶原样结构域、T4次要纤维蛋白或其三聚结构域(折叠)、胶原的C-前肽、表面活性剂蛋白A(SP-A)和甘露糖结合蛋白A(MBP-A)。30. The conjugate of any one of claims 26-29, wherein the first trimeric polypeptide of (b) is selected from adiponectin or its collagen-like domain, T4 minor fibrin, or Its trimerization domain (fold), the C-propeptide of collagen, surfactant protein A (SP-A) and mannose binding protein A (MBP-A).31.根据权利要求26-30中任一项所述的缀合物，其中(a)的所述三聚体多肽或(b)的所述第二三聚体多肽选自肿瘤坏死因子(TNF)超家族配体，任选地如前述权利要求中任一项或多项所定义的肿瘤坏死因子(TNF)超家族配体。31. The conjugate of any one of claims 26-30, wherein the trimeric polypeptide of (a) or the second trimeric polypeptide of (b) is selected from tumor necrosis factor (TNF) ) superfamily ligands, optionally tumor necrosis factor (TNF) superfamily ligands as defined in any one or more of the preceding claims.32.根据权利要求26-31中任一项所述的缀合物，其中对于(a)，所述六聚体多肽共价连接到所述三聚体多肽的N端，或者其中对于(b)，所述第一三聚体多肽共价连接到所述第二三聚体多肽的N端。32. The conjugate of any one of claims 26-31, wherein for (a), the hexameric polypeptide is covalently linked to the N-terminus of the trimeric polypeptide, or wherein for (b) ), the first trimeric polypeptide is covalently linked to the N-terminus of the second trimeric polypeptide.33.根据权利要求26-31中任一项所述的缀合物，其中对于(a)，所述六聚体多肽共价连接到所述三聚体多肽的C端，或者其中对于(b)，所述第一三聚体多肽共价连接到所述第二三聚体多肽的C端。33. The conjugate of any one of claims 26-31, wherein for (a), the hexameric polypeptide is covalently linked to the C-terminus of the trimeric polypeptide, or wherein for (b) ), the first trimeric polypeptide is covalently linked to the C-terminus of the second trimeric polypeptide.34.根据权利要求26-33中任一项所述的缀合物，其中对于(a)，所述六聚体多肽和所述三聚体多肽通过连接子分开，或者其中对于(b)，所述第一和第二三聚体多肽通过连接子分开，其中所述连接子任选地为生理稳定的连接子。34. The conjugate of any one of claims 26-33, wherein for (a) the hexameric polypeptide and the trimeric polypeptide are separated by a linker, or wherein for (b), The first and second trimeric polypeptides are separated by a linker, wherein the linker is optionally a physiologically stable linker.35.根据权利要求26-34中任一项所述的缀合物，其中所述缀合物是融合多肽。35. The conjugate of any one of claims 26-34, wherein the conjugate is a fusion polypeptide.36.根据权利要求26-35中任一项所述的缀合物，其中所述缀合物相对于单独的所述六聚体和/或三聚体多肽物理具有提升的物理、药代动力学和/或生物学性质。36. The conjugate of any one of claims 26-35, wherein the conjugate physically has improved physical, pharmacokinetics relative to the hexameric and/or trimeric polypeptide alone scientific and/or biological properties.37.根据权利要求36所述的缀合物，其中对于(a)，与所述六聚体多肽缀合使所述三聚体多肽的稳定性和/或血清半衰期相对于单独的所述三聚体多肽任选地增加约或至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、200％、300％、400％、500％、600％、700％、800％、900％或1000％或更多，或者其中对于(b)，与所述第一三聚体多肽缀合使所述第二三聚体多肽的稳定性和/或血清半衰期相对于单独的所述第二三聚体多肽任选地增加约或至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、200％、300％、400％、500％、600％、700％、800％、900％或1000％或更多。37. The conjugate of claim 36, wherein for (a), conjugation to the hexameric polypeptide increases the stability and/or serum half-life of the trimeric polypeptide relative to the trimeric polypeptide alone The polymeric polypeptide is optionally increased by about or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% or more, or wherein for (b), conjugation to the first trimeric polypeptide stabilizes the second trimeric polypeptide The sexual and/or serum half-life is optionally increased by about or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% or more.38.一种分离的多核苷酸，其编码根据权利要求1-37中任一项所述的缀合物，其中所述缀合物是融合蛋白，一种包括所述分离的多核苷酸的表达载体或一种包括所述分离的多核苷酸或所述表达载体的宿主细胞。38. An isolated polynucleotide encoding the conjugate of any one of claims 1-37, wherein the conjugate is a fusion protein, a polynucleotide comprising the isolated polynucleotide An expression vector or a host cell comprising the isolated polynucleotide or the expression vector.39.一种治疗组合物，其包括根据权利要求1-37中任一项所述的缀合物和药学上可接受的载剂或赋形剂。39. A therapeutic composition comprising the conjugate of any one of claims 1-37 and a pharmaceutically acceptable carrier or excipient.40.根据权利要求39所述的治疗组合物，其中所述缀合物形成六个ADI-TRAIL和/或TRAIL-ADI缀合物，任选地融合蛋白的六聚体复合物。40. The therapeutic composition of claim 39, wherein the conjugate forms a hexameric complex of six ADI-TRAIL and/or TRAIL-ADI conjugates, optionally fusion proteins.41.根据权利要求39或40所述的治疗组合物，其中所述缀合物的纯度为至少约95％且聚集度小于约5％，并且其中所述组合物基本上不含内毒素。41. The therapeutic composition of claim 39 or 40, wherein the conjugate is at least about 95% pure and has a degree of aggregation less than about 5%, and wherein the composition is substantially free of endotoxin.42.一种在有需要的受试者中治疗癌症、改善癌症症状或减少癌症进展的方法，其包括对所述受试者施用根据权利要求39-40中任一项所述的治疗组合物。42. A method of treating cancer, ameliorating cancer symptoms or reducing cancer progression in a subject in need thereof, comprising administering to the subject a therapeutic composition according to any one of claims 39-40 .43.根据权利要求42所述的方法，其中所述癌症选自以下中的一个或多个：肝细胞癌(HCC)、黑色素瘤、转移性黑色素瘤、胰腺癌、前列腺癌、小细胞肺癌、间皮瘤、淋巴细胞性白血病、慢性髓细胞性白血病、淋巴瘤、肝癌、肉瘤、白血病、急性髓性白血病、复发性急性髓性白血病、B细胞恶性肿瘤、乳腺癌、卵巢癌、结直肠癌、胃癌(gastric cancer)、胶质瘤(例如，星形细胞瘤、少突神经胶质瘤、室管膜瘤或脉络丛乳头状瘤)、多形性胶质母细胞瘤(例如，巨细胞胶质母细胞瘤或胶质肉瘤)、脑膜瘤、垂体腺瘤、前庭神经鞘瘤、原发性CNS淋巴瘤、原始神经外胚层肿瘤(成神经管细胞瘤)、非小细胞肺癌(NSCLC)、肾癌、膀胱癌、子宫癌、食道癌、脑癌、头颈癌、宫颈癌、睾丸癌和胃癌(stomach cancer)。43. The method of claim 42, wherein the cancer is selected from one or more of the following: hepatocellular carcinoma (HCC), melanoma, metastatic melanoma, pancreatic cancer, prostate cancer, small cell lung cancer, Mesothelioma, lymphocytic leukemia, chronic myeloid leukemia, lymphoma, liver cancer, sarcoma, leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia, B cell malignancies, breast cancer, ovarian cancer, colorectal cancer , gastric cancer, glioma (eg, astrocytoma, oligodendroglioma, ependymoma, or choroid plexus papilloma), glioblastoma multiforme (eg, giant cell glioblastoma or gliosarcoma), meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), non-small cell lung cancer (NSCLC) , kidney cancer, bladder cancer, uterine cancer, esophagus cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer and stomach cancer.